Ameliorating Nervous Systems Disorders

ABSTRACT

The present disclosure provides methods for the treatment of a mammal having a neurological condition, disease, or injury. The methods involve increasing the number of functional GABAergic interneurons at or near the site of the neurological disease, injury, or condition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit to U.S. provisional applicationSer. No. 61/050,980 filed on May 6, 2008, which application isincorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with the support of the United States governmentunder Contract number 5R01NS048528-04 by National Institutes of Health.The government has certain rights in the invention.

INTRODUCTION

Clinical management of conditions, diseases and injuries of the centraland peripheral nervous system usually focus of the prevention of furtherneurological damage or injury rather than on the repair or replacementof the damaged neurological tissue (e.g., neurons). For example,treatment of spinal cord injury includes the prevention of additionalspinal cord injury by physically stabilizing the spine through surgicaland non-surgical procedures and by inhibiting the inflammatory responsewith steroidal therapy. Thus, there remains a pressing need for improvedand effective treatments of the central and peripheral nervous systemthat are able to repair or replace damaged or injured neural tissue.

SUMMARY

In some embodiments, a method is provided for the treatment of a mammalhaving a neurological condition, disease, or injury comprisingincreasing the number of functional GABAergic interneurons at or nearthe site of the neurological disease, injury, or condition, wherein thefunctional GABAergic interneurons functionally integrate with endogenousneurons and restore balance to neuronal circuitry that is dysregulatedin neurological conditions, diseases, or injuries. In some embodiments,the increase in the functional GABAergic interneurons is bytransplantation using injections of MGE (medial ganglionic eminence)precursor cells. In some embodiments, the MGE precursor cells are ableto migrate at least 0.5 mm from the transplantation site.

In some embodiments, the neurological condition, disease, or injury is adegenerative disease, genetic disease, acute injury, or chronic injury.In some embodiments, the neurological condition, disease, or injurycomprises Parkinson's disease, epilepsy, spasticity, multiple sclerosis,stroke, spinal cord injury, brain injury, or chronic pain disorders.

In some embodiments, the neurological condition is epilepsy, whereintransplantation of MGE precursor cells result in at least a 10%reduction in spontaneous electrographic seizure activity. In someembodiments, the neurological condition is epilepsy, whereintransplantation of MGE precursor cells result in at least a 10%reduction in seizure duration. In some embodiments, the neurologicalcondition is epilepsy, wherein transplantation of MGE precursor cellsresult in at least a 10% reduction in seizure frequency. In someembodiments, the neurological condition is epilepsy, whereintransplantation of MGE precursor cells result in at least a 10%reduction in required antiepileptic drug use.

In some embodiments, the neurological disease is Parkinson's disease,wherein transplantation of MGE precursor cells result in at least a 10%reduction in required anti-Parkinsonian drug use. In some embodiments,the neurological disease is Parkinson's disease, wherein transplantationof MGE precursor cells result in at least a 10% reduction in tremor atrest, rigidity, akinesia, bradykinesia, postural instability, flexedposture and/or freezing. In some embodiments, the neurological diseaseis Parkinson's disease, wherein the MGE cells transplanted into thestriatum survive for at least 6 months.

In some embodiments, the neurological condition is spasticity, whereintransplantation of MGE precursor cells mitigates or obviates the needfor intrathecal medication or surgery. In some embodiments, theneurological condition is spasticity, wherein transplantation of MGEprecursor cells result in at least a 10% reduction in requiredantispasmodic drug use

In some embodiments, the MGE precursor cells express a therapeuticprotein or peptide, or neurotransmitter. In some embodiments, thetherapeutic protein or peptide comprises a neurotrophin, a neuropoieticcytokine, a fibroblast growth factor (e.g., acidic and basic FGF), aninhibitory growth factor, or a cytokine useful in the treatment ofinfectious disease, brain tumors, or brain metastases.

In some embodiments, the MGE precursor cells are injected into thestriatum, basal ganglia, dorsal ganglia, ventral horn, or lumbar theca.In some embodiments, the mammal does not require immunosuppressivetherapy following transplantation.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference. Nothing citedherein is to be construed as an admission that the invention is notentitled to antedate such disclosure by virtue of prior invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a collection of schematics as follows: Panel A. Experimentaldesign. 6-OHDA-surgery was performed on day 1 and behavioral tests onweeks 3 and 5. If the rat showed parkinsonian behavior, it was injectedwith MGE cells on week 6. The behavioral tests were repeated on weeks 9,11, 14 and 18. Panel B. Unilateral lesion of the rat nigrostriatalprojection. Tyrosine hydroxylase (TH) labels dopaminegic cells. Theipsilateral side to the injection did not show any stain for TH, whilethe contralateral side had numerous TH+ cells in the SNc, after 2 weeks.Panel C. Drug-induced rotations were measured in an automated rotometerbowl. After intraperitoneal injection of apomorphine, the animals werefitted with a jacket that was attached via a cable to a rotation sensor,the animals were placed into the test bowl and the number of clockwiseor counterclockwise rotations was recorded over a test period of 40minutes. Panel D. Rats were placed into a runway. The floor of therunway was covered with paper. The rats were trained to run down therunway. At the start of each test, the animals' feet were dipped inblack ink before being placed at the beginning of the runway. Panel E.The length of stride was measure for each test. 6-OHDA length of stridewas shorter than that of controls. Panel F. 6-OHDA rats tended to wanderfrom side to side as they walked down the runway rather than follow astraight path. Scale bar: A=30 μm.

FIG. 2 is a collection of images as follows: Panel A. Striatum 4 weeksafter transplant. The transplanted GFP-MGE cells migrated 2 to 2.5 mm inall the directions from the site of injection. The cells covered thewhole span of the striatum but never migrated outside it. Panel B.Transplanted cells 8 weeks after transplant had a fully matureappearance, an oblong body shape and numerous processes that extended upto 50 μm, and occupied the whole striatum even in areas far away fromcellular GFP-MGE somas. The processes intermingled and seemed to contactone another. Scale bars: A=0.5 mm; B=25 μm.

FIG. 3 is a collection of images as follow, illustrating that most (3:1)MGE cells showed interneuronal fate when transplanted into the adultstriatum. Four weeks after transplant, MGE-cells expressed the matureneuronal markers NeuN+(A, 75±6%), GAD (B, 60±11%), GABA+(C, 75±4%), GAT(D, 50±9%), CB (E, 24±7%), CR (F, 8.3±0.5%), Substance P (G, 6±2%),CNPase (H, 25%±4), Somatostatin (I, 1±0.5%) and Chat. Scale bars: B1=10μm and applies to Panels A1, C1, D1, E1, F1, G1, H1, I1, and J1; A3=30μm and applies to Panels A2, B1, B2, C1, C2, D1, D2, E1, E2, F1, F2, G1,G2, H1, H2, I1, I2, J1, and J2.

FIG. 4 is a collection of graphs and images showing that MGE cellstransplanted into the striatum retain the basic membrane properties thatare characteristics of cortical interneurons Panel A. 67% of the MGEcells expressed synaptophysin in their processes (white arrows). PanelB. Alexa-594 was included in the fill solution of the glassmicroelectrode to determine whether the obtained whole-cell recordingswere from the targeted GFP-expressing MGE transplant cells. Panel C. MGEcells fired repetitive non-accomodating action potentials with largeafterhyperpolarizations when stimulated with a series of depolarizingcurrents, as is common for MGE derived interneurons. Panel D. Some ofthe transplanted MGE cells (n=2/13) did not possess neuronal membraneproperties and did not fire action potentials. Panel E. MGE transplantcells fired spontaneous action potentials, and Panel F. exhibitedspontaneous synaptic currents at a frequency of 1.1 psc/s, which wassimilar to that recorded from host cells (1.0 psc/s). Scale bars: A=3μm; B=5 μm.

FIG. 5 is a collection of graphs as follows: Panel A. Rotation test.6-OHDA rats (n=21) performed far more rotations than control rats (n=16)before receiving an MGE cell transplant. Upon transplantation, however,they started performing significantly fewer contra-lateral turns thannon-transplanted 6-OHDA controls (n=12). This trend continued until thelast time point (week 18). Sham-transplanted 6-OHDA rats (n=11)performed similarly to non-transplanted 6-OHDA rats (n=12). Panel B.Length of stride test. 6-OHDA rats (n=12) had an average stride lengthof 13.2±0.2 cm, significantly shorter than the average stride length ofcontrol rats (15.1±0.3, n=16). Upon MGE cells transplantation, thestride length of 6-OHDA rats increased and eventually reached the samevalue as in control rats (15.5±0.4 cm, n=21). The stride length of6-OHDA rats that had received a sham transplant (n=11) was notsignificantly different from that of 6-OHDA rats (n=12). Panel C. Widthof path test. 6-OHDA rats had a width of path of 3.4±0.3 cm (n=12),significantly different from the 2.5±0.3 cm width of path of controlrats (n=16). Upon MGE cells transplantation, the path width decreasedand eventually reached control value (n=16). The path width of shamtransplanted 6-OHDA rats (n=11) was not significantly different fromthat of 6-OHDA rats (n=12).

FIG. 6 is a collection of images showing MGE transplanted cellsexpressed Dopamine Receptors 1 (DR1, A1-A2) and Dopamine Receptor 2(DR2, B1-B2). DR1 and DR2 are present in somatic membrane and processesof MGE cells. Panels A2 and A3. High magnification images of MGE cellsthat expressed DR1 (A2) and DR2 (B2). Panel A1=30 μm and applies toPanel B1; Panel A2=3 μm and applies to Panel B2.

FIG. 7 is a collection of graphs showing the following: Panel A. Thelength of stride was significantly higher in control rats injected withMGE cells (n=6) than in control rats (n=16). Panel B. Open field testshow an increase activity in wild-type MGE transplanted rats compared tocontrol rats. Panel C. Representative open field zone map. Wild-type MGEtransplanted rat has a major level of motor activity when compared to acontrol rat.

FIG. 8 is a collection of images showing MGE cells transplanted into theSTN survived but did not migrate from the site of injection (Panel A).None of the MGE cells in the STN expressed the neuronal marker NeuN(Panel B), the inhibitory interneuron markers GABA (Panel E) or GAD(Panel G), or the calcium sequestering proteins CR (Panel H) or CB(Panel I). MGE cells transplanted STN expressed GFAP (75±5%, Panels Cand J) or the oligodendrocyte marker CNPase (30±9%, Panels D and K). Asmall percentage of MGE cells in the STN expressed the GABA transporterGAT1 (13±5%, Panel F). Scale bars: A=25 μm; I=25 μm and applies toPanels B, C, D, E, F, G and H; K=5 μm and applies to J.

FIG. 9 is a collection of schematics and images showing the following:Panel A generally illustrates the transplantation of MGE cells fromE13.5 embryonic GFP+ transgenic donor into mice expressing greenfluorescent protein (GFP) on postnatal day 2 (P2); bilateral injectionswere made. MGE cells are then analyzed 30 days after transplantation (30DAT). Panel B illustrates transplanted MGE cells, harvested from miceexpressing GFP at 30 DAT and co-labeled with antibodies to GABA, GAD67,calretinin (CR), parvalbumin (PV), neuropeptide Y (NPY), andsomatostatin (SOM). Panels C and D illustrate that the MGE-GFP neuronsexhibit firing properties similar to endogenous interneuron sub-typessuch as fast-spiking (FS), regular-spiking non-pyramidal cells (RSNP),and stuttering (STUT). The plot in 1d summarizes all GFP-positive cellsthat were recorded; one cell had non-firing properties similar to anastrocyte (astro).

FIG. 10 is a collection of graphs and images as follows: Panel A showsan interneuron, identified by their oval and often multi-polarmorphologies under IR-DIC. Panel B confirms post hoc that theinterneurons were correctly identified with biocytin labeling. Panel Cshows GABA-mediated current from control (un-transplanted) and graftedinterneurons that were voltage-clamped in bath solution containingglutamate receptor antagonists (DNQX and APV). Panels D-F show nosignificant differences in GABA-mediated inhibition onto endogenousinterneurons between control and grafted mice at 30 DAT using IPSCanalysis.

FIG. 11 shows that white matter stimulation elicited IPSCs on LayerII/III pyramidal neurons in regions containing GFP+ cells with kineticproperties that were not different between control and grafted animals.An exemplary graph is shown in Panel A, and the data presented ingraphic form in Panel B.

FIG. 12 is a collection of images and graphs as follows: Panel A,subpanels 1-4 show that early postnatal bilateral transplantation ofMGE-GFP progenitors generated new GFP-immunoreactive (IR) cells in hostneocortex with distributions between 0.75 and 5 mm from injection site.Panel A, subpanel 5 show that a threshold of ˜40,000 GFP—IR cells peranimal, with migration≧0.5 mm from the injection site, was defined as asuccessful graft. Panel B illustrates confirmation of high-amplitudesynchronous electrographic seizure activity using scalpelectroencephalographic (EEG) recording. Panel C, graph 1 shows thepercentage observed for behavioral seizure manifestations includingforelimb clonus (Stage 3, S3) in control mice and in mice withphenobarbital (PB), carbamezepine (CBZ), MGE, and phenyloin (PHT). Formice grafted with MGE cells at P2, the percentage of animals exhibitingS3 seizures was reduced to ˜50%. Panel C, graph 2 shows the latency tofirst seizure behavior for control mice and in mice with phenobarbital(PB), carbamezepine (CBZ), MGE, and phenyloin (PHT). For mice graftedwith MGE cells at P2, the latency to first seizure behavior wasunchanged from that of the control mice.

FIG. 13 is a collection of graphs as follows: Panel A showsvideo-electroencephalography (EEG) used to examine MGE transplanteffects on spontaneous seizures in mice epilepsy model. Panel B showsthe duration of the spontaneous seizures in mice epilepsy models withMGE transplants. Panel C shows the frequency of the spontaneous seizuresin mice epilepsy models with MGE transplants. Panel D shows theKaplan-Maier survival plots for postnatal days. Panel E shows thepercentage of mice that survive at P55. Panel F shows new GFP celldensity per brain in Kv1.1−/− mice that survive at P57.

DETAILED DESCRIPTION

While exemplary embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

The practice of the present methods will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare described in the literature. See, for example, Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984);Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D.Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D.Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I.Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRLPress, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984);the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); GeneTransfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154and 155 (Wu et al. eds.), Immunochemical Methods In Cell And MolecularBiology (Mayer and Walker, eds., Academic Press, London, 1987); HandbookOf Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “acell” includes a plurality of such cells and so forth. Unless definedotherwise, all technical and scientific terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art towhich this invention belongs. Although any materials and methods similaror equivalent to those described herein can be used to practice or testthe present invention, exemplary materials and methods are nowdescribed.

As common methods of administering the MGE precursor cells of thepresent disclosure to animals, particularly humans, which are describedin detail herein, include injection or implantation of the MGEprecursors cells into target sites in the animals, the cells of thedisclosure can be inserted into a delivery device which facilitatesintroduction by, injection or implantation, of the cells into theanimals. Such delivery devices include tubes, eg., catheters, forinjecting cells and fluids into the body of a recipient animal. In apreferred embodiment, the tubes additionally have a needle, eg., asyringe, through which the cells can be introduced into the animal at adesired location. The MGE precursor cells can be inserted into such adelivery device, eg., a syringe, in different forms. For example, thecells can be suspended in a solution or embedded in a support matrixwhen contained in such a delivery device. As used herein, the term“solution” includes a pharmaceutically acceptable carrier or diluent inwhich the cells remain viable. Pharmaceutically acceptable carriers anddiluents include saline, aqueous buffer solutions, solvents and/ordispersion media. The use of such carriers and diluents is well known inthe art. The solution is preferably sterile and fluid to the extent thateasy syringability exists. Preferably, the solution is stable under theconditions of manufacture and storage and preserved against thecontaminating action of microorganisms such as bacteria and fungithrough the use of, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. Solutions of the presentdisclosure can be prepared as described herein in as a pharmaceuticallyacceptable carrier or diluent and, as required, other ingredientsenumerated above, followed by filter sterilization.

The present disclosure also provides substantially pure MGE precursorcells that can be used therapeutically for treatment of variousdisorders. To illustrate, the MGE precursors of the disclosure can beused in the treatment or prophylaxis of a variety of conditions,diseases, or disorders. For instance, the MGE precursors can be used toproduce populations of differentiated neurons for repair, replacement,or amelioration of damaged nervous system tissue for the treatment orprophylaxis of Parkinson's disease, epilepsy, schizophrenia, chronicpain disorders, neuropathic pain, multiple sclerosis, neuropathy, damagefrom traumatic injury, stroke, or ischemia and the like.

The invention will now be further described by way of reference only tothe following non-limiting examples. It should be understood, however,that the examples following are illustrative only, and should not betaken in any way as a restriction on the generality of the inventiondescribed above. In particular, while the methods and compositions ofthe present disclosure are described in detail in relation to the use ofmouse and rat cells, it will be clearly understood that the findingsherein are not limited to these-types of cells, but would be usefulgrowing any MGE precursor cell from any animal, including humans.

When the cells are implanted into the brain, stereotaxic methods willgenerally be used as described in Leksell and Jernberg, Acta Neurochir.,52:1-7 (1980) and Leksell et al., J. Neurosurg., 66:626-629 (1987), bothof which are incorporated herein by reference. Localization of targetregions will generally include pre-implantation MRI as described inLeksell et al., J. Neurol. Neurosurg. Psychiatry, 48:14-18 (1985),incorporated herein by reference. Target coordinates will be determinedfrom the pre-implantation MRI.

Prior to implantation, the viability of the cells may be assessed asdescribed by Brundin et al., Brain Res., 331:251-259 (1985),incorporated herein by reference. Briefly, sample aliquots of the cellsuspension (1-4 μl) are mixed on a glass slide with 10 μl of a mixtureof acridine orange and ethidium bromide (3.4 g/ml of each component in0.9% saline; Sigma). The suspension is transferred to a hemocytometer,and viable and non-viable cells were visually counted using afluorescence microscope under epi-illumination at 390 nm combined withwhite light trans-illumination to visualize the counting chamber grid.Acridine orange stains live nuclei green, whereas ethidium bromide willenter dead cells resulting in orange-red fluorescence. Cell suspensionsshould generally contain more than about 98% viable cells.

In humans, injections will generally be made with sterilized 10 μlHamilton syringes having 23-27 gauge needles. The syringe, loaded withcells, is mounted directly into the head of a stereotaxic frame. Theinjection needle is lowered to predetermined coordinates through smallburr holes in the cranium, 40-50 μl of suspension are deposited at therate of about 1-2 μl/minute and a further 2-5 minutes are allowed fordiffusion prior to slow retraction of the needle. Frequently, two ormore separate deposits will be made, separated by 1-3 mm, along the sameneedle penetration, and up to 5 deposits scattered over the target areacan readily be made in the same operation. The injection may beperformed manually or by an infusion pump. At the completion of surgeryfollowing retraction of the needle, the patient is removed from theframe and the wound is sutured. Prophylactic antibiotics orimmunosuppressive therapy may be administered as needed.

In some embodiments of the present disclosure, the implanted cells maybe transfected with a DNA sequence encoding a peptide. The peptide maybe an enzyme which catalyzes the production of a therapeutic compoundincluding the production of a neurotransmitter, e.g., the DNA couldencode tyrosine hydroxylase which catalyzes the synthesis of dopaminethat is effective in the treatment of Parkinsonism. The DNA may alsoencode a neurotrophic factor. Useful neurotrophic factors include theneurotrophins (e.g., NGF; brain-derived neurotrophic factor, BDNF; andneurotrophins NT-3 and NT-4/5); the neuropoietic cytokines (e.g.,ciliary neurotrophic factor, CNTF); and the fibroblast growth factors(e.g., acidic and basic FGF). The DNA may also encode an inhibitorygrowth factor, or a cytokine useful in the treatment of infectiousdisease, brain tumors, or brain metastases.

Generally, the DNA sequence will be operably linked to a transcriptionalpromoter and a transcriptional terminator. The DNA sequence may also belinked to a transcriptional enhancer. Expression of the DNA in theimplanted cells may be constitutive or inducible. A variety ofexpression vectors having these characteristics may carry the DNA fortransfection of the cells, such as plasmid vectors pTK2, pHyg, andpRSVneo, simian virus 40 vectors, bovine papillomavirus vectors orEpstein-Barr virus vectors, as described in Sambrook et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, 1988, previouslyincorporated herein by reference. The vectors may be introduced into thecells by standard methods, such as electroporation, calciumphosphate-mediated transfection, polybrene transfection, and the like.

In some embodiments, the present disclosure is useful in the treatmentof degenerative diseases. A degenerative disease is a disease in whichthe decline (e.g., function, structure, biochemistry) of particular celltype, e.g., neuronal, results in an adverse clinical condition. Forexample, Parkinson's disease is a degenerative disease in the centralnervous system, e.g., basal ganglia, which is characterized byrhythmical muscular tremors, rigidity of movement, festination, droopyposture and masklike facies. Degenerative diseases that can be treatedwith the substantially homogenous cell populations of the presentdisclosure include, for example, Parkinson's disease, multiplesclerosis, epilepsy, Huntington's, dystonia, (dystonia musculmusculorumdeformans) and choreoathetosis.

In some embodiments, the present disclosure is useful in the treatmentof conditions caused by an acute injury. An acute injury condition is acondition in which an event or multiple events results in an adverseclinical condition. The event which results in the acute injurycondition can be an external event such as blunt force or compression oran internal event such as sudden ischemia (e.g., stroke or heartattack). Acute injury conditions that can be treated with thesubstantially homogenous cell populations of the present disclosureinclude, for example, spinal cord injury, traumatic brain injury, braindamage resulting from myocardial infarction and stroke.

In some embodiments, the present disclosure provides methods of treatinga human suffering from a neurological condition, comprising the step ofadministering to the human a substantially homogenous cell population ofthe present disclosure. “A neurological condition,” as used herein,refers to any state of the nervous system (central or peripheral nervoussystem) which deviates in any manner from a normal nervous system ornervous system of a mammal, e.g., human, not affected by a neurologicalcondition. The neurological condition can be a condition of the central(brain or spinal cord) or peripheral nervous system. The neurologicalcondition can be, for example, the result or consequence of a disease,e.g., Parkinson's disease or multiple sclerosis, acute injury condition,e.g., stroke, brain injury, spinal cord injury, or a combination ofdisease and acute injury condition. Other neurological conditions whichcan be treated with the substantially homogenous population of cells ofthe present disclosure include, for example, chronic or intractablepain, primary brain tumors, or metastasizes.

In some embodiments, the administered cells comprise a substantiallyhomogenous population. In some embodiments, the substantially homogenouspopulation comprises cells wherein at least 25% of the cells become GABAexpressing cells. In some embodiments, the substantially homogenouspopulation comprises cells wherein at least 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or 99% of the cells become GABA expressing cells. In someembodiments, at least 25% of the cells comprising the substantiallyhomogenous population of cells migrate at least 0.5 mm from theinjection site. In some embodiments, at least 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or 99% of the cells comprising the substantiallyhomogenous population of cells migrate at least 0.5 mm from theinjection site. In some embodiments, the majority of the cellscomprising the substantially homogenous population of cells migrate atleast 1.0, 1.5, 2.0, 3.0, 4.0, or 5.0 mm from the injection site. Insome embodiments, at least 25% of the substantially homogenouspopulation of cells becomes functionally GABAergic interneurons. In someembodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% ofthe cells become functionally GABAergic interneurons. In someembodiments, at least 25% of the substantially homogenous population ofcells becomes functionally GABAergic interneurons that integrate withendogenous neurons. In some embodiments, at least 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, or 99% of the substantially homogenous population ofcells become functionally GABAergic interneurons that integrate withendogenous neurons.

In some embodiments, the substantially homogeneous population of cellsbecomes functional GABAergic interneurons and express one or more of thefollowing: parvalbumin, calbindin, somatostatin, calretinin,neuropeptide Y, nitric oxide synthase, ChAT, NADPH diaphorase.

In some embodiments, the implanted functionally GABAergic interneuronsincrease the overall level of GABA-mediated synaptic inhibition in thehost brain by at least 5%. In some embodiments, the implantedfunctionally GABAergic interneurons increase overall level ofGABA-mediated synaptic inhibition in the host brain by at least 10%,15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. The increasedinhibition can be monitored in patients by assaying for GABA levels inregions containing transplanted MGE cells.

In some embodiments, the functionally integrated transplantedinterneurons represent at least 1% of the total number of the nativestriatal interneurons. In some embodiments, the functionally integratedtransplanted interneurons represent at least 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 12%, 15%, 20%, 25%, or 30% of the total number of the striatalinterneurons.

In some embodiments, the substantially homogenous cells secrete at leastone neurotransmitter, therapeutic factor, cytokine and/or trophicfactor. In some embodiments, the synthesis and secretion of at least onetherapeutic factor, cytokine and/or trophic factor from thesubstantially homogenous population of cells of the present disclosurecan protect surrounding cells near or distant from the site oftransplantation from further damage as a consequence of thedegenerative, acute injury or neurological condition. In someembodiments, the synthesis and secretion of at least one therapeuticfactor, cytokine and/or trophic factor from the substantially homogenouspopulation of cells of the present disclosure can also, oralternatively, promote regeneration of cells and tissues of the host,e.g., human suffering from a degenerative condition, acute injury, orneurological condition.

In some embodiments, the cell populations of the present disclosure havethe capacity to respond to intrinsic signals, e.g., at the sites oftransplantation, during migration, or at final migratory location, andexogenous cues to differentiate into neurons. The cell populations ofthe present disclosure can provide a readily available source of cellsfor use in treating animals, preferably mammals, and most preferablyhumans.

“MGE progenitor cell” or “MGE precursor cell,” as used herein, refers toa cell obtained from a source, e.g., embryonic medial ganglioniceminence, umbilical cord blood, adult bone marrow, fat, skin, etc., thathas substantially similar characteristics of a cell isolated from theMGE of E13.5 mouse fetuses, E14.5 rat fetuses, or develops into a cellwith the substantially similar characteristics of a cell isolated fromthe MGE of E14.5 mouse fetuses. MGE progenitor cells can be isolatedfrom human fetuses at 12-14 gestational weeks. A MGE progenitor cell canbe a cell isolated from central nervous system tissue that issubstantially similar to the MGE of E13.5 mice fetus or E14.5 rat fetus,a primary culture of cells grown from cells isolated from centralnervous system tissue that is substantially similar to the MGE of E13.5mice, E14.5 rat, or cells generated from other sources such as celllines derived from embryonic or adult stem cells that are furtherdifferentiated to have substantially similar characteristics of cellsisolated from the MGE of E13.5 mice or E14.5 rat.

In some embodiments, the cell line is produced by regulating theproduction of telomerase. In some embodiments, telomerase expression isforced by the expression of exogenous hTERT as detailed in Ouellette, M.M. et al. The establishment of telomerase-immortalized cell linesrepresenting human chromosome instability syndromes. Human MolecularGenetics, 9:403-411, 2000. In some embodiments, the cell line is derivedfrom pluripotent stem cells that are derived following reprogramming ofadult somatic cells by somatic cell nuclear transfer as detailed by I.Wilmut, et al. Nature, 385, 810, 1997. In some embodiments, the cellline is derived from pluripotent stem cells that were derived followingreprogramming of adult somatic cells by fusion with embryonic stem cellsas described by Cowan, C. et al. Nuclear reprogramming of somatic cellsafter fusion with human embryonic stem cells. Science, 309, 1369-1372,2005. In some embodiments, the cell line is derived from pluripotentstem cells that were derived following reprogramming of adult somaticcells by exposure to a defined set of reprogramming factors. In someembodiments, the reprogramming factors are OCT4, SOX2, NANOG, and LIN28as detailed by Yu, J., et al. Induced pluripotent stem cell linesderived from human somatic cells. Science, 318, 1917-1920, 2007. In someembodiments, the reprogramming factors are Oct3/4, Sox2, Klf4, and c-Mycas detailed by Takahashi, K., et al. Induction of pluripotent stem cellsfrom adult human fibroblasts. Cell, 131, 861-871, 2007.

Selected cells can be used directly from cultures or stored for futureuse, e.g., by cryopreserving in liquid nitrogen. If cyropreserved, MGEprecursors must be initially thawed before placing the MGE precursors ina transplantation medium. Methods of freezing and thawing cryopreservedmaterials such that they are active after the thawing process arewell-known to those of ordinary skill in the art.

In some embodiments, the present disclosure includes a pharmaceuticalcomposition comprising a substantially homogeneous cell population ofMGE precursors. In some embodiments, the pharmaceutical composition hasat least about 10⁵ substantially homogeneous cells. In some embodiments,the pharmaceutical composition has at least about 10⁶, 10⁷, 10⁸, 10⁹, or10¹⁰ substantially homogeneous cells. The cells comprising thepharmaceutical composition can also express at least oneneurotransmitter, neurotrophic factor, inhibitory factor, or cytokine.

The cells of the present disclosure can be, for example, transplanted orplaced in the central, e.g., brain or spinal cord, or peripheral nervoussystem. The site of placement in the nervous system for the cells of thepresent disclosure is determined based on the particular neurologicalcondition, e.g., direct injection into the lesioned striatum, spinalcord parenchyma, or dorsal ganglia. For example, cells of the presentdisclosure can be placed in or near the striatum of patients sufferingfrom Parkinson's disease. Similarly, cells of the present disclosure canbe placed in or near the spinal cord (e.g., cervical, thoracic, lumbaror sacral) of patients suffering from a spinal cord injury. One skilledin the art would be able to determine the manner (e.g., needle injectionor placement, more invasive surgery) most suitable for placement of thecells depending upon the location of the neurological condition and themedical condition of the patient.

The substantially homogenous cells of the present disclosure can beadministered alone or as admixtures with conventional excipients, forexample, pharmaceutically, or physiologically, acceptable organic, orinorganic carrier substances suitable for enteral or parenteralapplication which do not deleteriously react with the cells of thepresent disclosure. Suitable pharmaceutically acceptable carriersinclude water, salt solutions (such as Ringer's solution), alcohols,oils, gelatins and carbohydrates such as lactose, amylose or starch,fatty acid esters, hydroxymethycellulose, and polyvinyl pyrrolidine.Such preparations can be sterilized and, if desired, mixed withauxiliary agents such as lubricants, preservatives, stabilizers, wettingagents, emulsifiers, salts for influencing osmotic pressure, buffers,coloring, and/or aromatic substances and the like which do notdeleteriously react with the cells of the present disclosure.

When parenteral application is needed or desired, particularly suitableadmixtures for the cells are injectable, sterile solutions, preferablyoily or aqueous solutions, as well as suspensions, emulsions, orimplants. In particular, carriers for parenteral administration includeaqueous solutions of dextrose, saline, pure water, ethanol, glycerol,propylene glycol, peanut oil, sesame oil and polyoxyethylene-blockpolymers. Pharmaceutical admixtures suitable for use in the presentdisclosure are well-known to those of skill in the art and aredescribed, for example, in Pharmaceutical Sciences (17th Ed., Mack Pub.Co., Easton, Pa.) and WO 96/05309 the teachings of both of which arehereby incorporated by reference.

The substantially homogenous population of cells can be used alone or incombination with other therapies when administered to a human sufferingfrom a neurological condition. For example, steroids or pharmaceuticalsynthetic drugs can be co-administered with the cells of the presentdisclosure. Likewise, treatment of spinal cord injury can include theadministration/transplantation of the cells of the present disclosure ina human whose spine has been physically stabilized.

The dosage and frequency (single or multiple doses) of theadministration or transplantation of the cells to a human, including theactual number of cells transplanted into the human, can vary dependingupon a variety of factors, including the particular condition beingtreated, e.g., degenerative condition, acute injury, neurologicalcondition; size; age; sex; health; body weight; body mass index; diet;nature and extent of symptoms of the neurological condition beingtreated, e.g., early onset Parkinson's disease versus advancedParkinson's disease; spinal cord trauma versus partial or completesevering of the spinal cord); kind of concurrent treatment, e.g.,steroids; complications from the neurological condition; extent oftolerance to the treatment or other health-related problems. Humans witha degenerative condition, acute injury, or neurological condition can betreated of once or repeatedly with cells of the present disclosure,e.g., about 10⁶ cells, at the same or different site. Treatment can beperformed monthly, every six months, yearly, biannually, every 5, 10, or15 years, or any other appropriate time period as deemed medicallynecessary.

The methods of the present disclosure can be employed to treatneurological conditions in mammals other than human mammals. Forexample, a non-human mammal in need of veterinary treatment, e.g.,companion animals (e.g., dogs, cats), farm animals (e.g., cows, sheep,pigs, horses) and laboratory animals (e.g., rats, mice, guinea pigs).

Parkinson's Disease

Parkinson's disease (PD) affects approximately 150 per 100,000 people inthe United States and Europe. PD is characterized by motor impairment aswell as cognitive and autonomic dysfunction and disturbances in mood.Four cardinal features of PD can be grouped under the acronym TRAP:Tremor at rest, Rigidity, Akinesia (or bradykinesia) and Posturalinstability. In addition, flexed posture and freezing (motor blocks)have been included among classic features of parkinsonism, with PD asthe most common form. Existing treatments can attenuate the symptoms ofPD but there is no cure. The motor symptoms of PD result primarily fromthe loss of dopamine containing neurons in the substantia nigra compacta(SNc) that extend axonal projections to the striatum and releasedopamine (for review see (Litvan et al., 2007). The SNc and the striatumbelong to the basal ganglia, a network of nuclei which integrateinhibitory and excitatory signals to control movement. Loss of SNc cellsin PD reduces the amount of dopamine release into the striatum,producing a neurotransmitter imbalance that inhibits the output of thebasal ganglia and produces hypokinetic signs (for review (DeLong andWichmann, 2007).

The striatum is composed of three classes of neurons. Medium spinyneurons are the projection neurons of the striatum and account for 95%of the total number of striatal neurons. The medium spiny neurons areGABAergic, express calbindin and substance P, give rise to nearly alloutputs from the striatum, and receive nearly all the synapses frombrain structures that project into the striatum (Tepper and Bolam,2004). A second class of neuron is the large cholinergic neuron or largespiny neuron that is an excitatory striatal interneuron that expressesthe marker cholineacetyltransferase (Chat). Cholinergic interneuronsmodulate the sub- and supra-threshold responses of the medium spinyneurons to cortical and/or thalamic inputs (Tepper and Bolam, 2004). Thethird class of striatal neuron is the GABAergic inhibitory interneuronor small spiny neuron of the striatum. These cells account forapproximately 1-2% of the total number of striatal neurons. There arethree subtypes of GABAergic inhibitory interneuron, and each subtype canbe identified by the co-expression of specific markers: GABAergicinterneurons that express calretinin (CR); GABAergic interneurons thatexpress parvalbumin (PV); and GABAergic interneurons that expresssomatostatin, NADPH-diaphorase, and NOS (Kawaguchi et al., 1995; Tepperand Bolam, 2004). The GABAergic interneurons are the main source ofinhibition of medium spiny neurons (Koos and Tepper, 1999; Koos et al.,2004; Plenz and Kitai, 1998). Each of the three types of GABAergicinterneurons produce a strong inhibitory postsynaptic potential inmedium spiny neurons, the function of which is to influence the precisetiming of action potential firing in either individual or ensembles ofmedium spiny neurons. Both excitatory and inhibitory striatalinterneurons are important sites of action for neuromodulators in theneostriatum, and act in different but complementary ways to modify theactivity of the medium spiny projection neurons (Tepper and Bolam,2004).

Striatal projection neurons and interneurons originate from theembryonic primordium of the basal ganglia, the ganglionic eminences.Inhibitory projection neurons are believed to derive from the medialganglionic eminence (MGE, (Anderson et al., 1997; Deacon et al., 1994;Olsson et al., 1995), and the cholinergic excitatory projection neuronsare thought to derive from the lateral ganglionic eminence (LGE) (Olssonet al., 1998). Previous data indicate that the GABAergic interneuronsmay have mixed origins. The CR+ subclass of interneurons derives mostlyfrom MGE, but as many as ten percent may be derived from the LGE (Marinet al., 2000). The PV+ subclass of interneurons is thought to deriveentirely from MGE (Marin et al., 2000). Transplantation studies suggestthat Som+ interneurons may originate from both the LGE and MGE (Olssonet al., 1998), although the expression pattern of the transcriptionfactor Nkx2.1, which is required for the specification of MGE derivates,suggests that Som+ cells are derived only from the MGE (Marin et al.,2000). The embryonic MGE also produces a substantial number ofneocortical interneurons that migrate long distances over a tangentialpathway to the dorsal neocortex, where they mature into local circuitGABAergic interneurons (Anderson et al., 1999; Lavdas et al., 1999;Wichterle et al., 1999). MGE cells retain the capacity for dispersal andintegration when grafted heterochronically into neonatal or adult brain(Grasbon-Frodl et al., 1997; Olsson et al., 1997; Wichterle et al.,1999), develop into mature neurons when re-transplanted into theembryonic MGE (Butt et al., 2005), and can significantly increase thelevels of inhibition exerted on neocortical projection neurons whengrafted into the neocortex (Alvarez-Dolado et al., 2006).

The most widely used treatment for PD is administration of the dopamineprecursor, levodopa, which improves motor behavior but also producesundesirable side effects including dyskinesias. Surgical approaches havebeen developed that involve electrical stimulation or ablation of themotor thalamus, the subthalamic nucleus, or the globus pallidus.Additional therapeutic strategies have been based on restoring orincreasing concentrations of dopamine in the basal ganglia throughtransplantation of adult or embryonic dopamine-releasing tissues or stemcells.

To treat the motor symptoms of Parkinson's disease produced by areduction of dopaminergic input, a non-dopamine based strategy thatmodified the circuit activity in the basal ganglia was employed. MGEcells were transplanted into the striatum of rats treated with6-hydroxydopamine (6-OHDA), a well-established model of PD. Thistreatment relies on the ability of MGE cells to migrate, functionallyintegrate, and increase levels of inhibition in the host brain aftertransplantation. Transplanted MGE cells migrated from the site ofinjection and dispersed throughout the host striatum. Most MGEtransplant cells acquired a mature neuronal phenotype and expressedneuronal and GABAergic markers. In addition, the transplanted cellsexpressed a variety of markers that are characteristic of striatalGABAergic interneurons such as CB, CR, CB, and Som. Finally, the MGEtransplant cells became physiologically mature, integrated into the hostcircuitry, and improved the motor symptoms of PD in the rat 6-OHDAmodel. These results indicate that the transplantation of GABAergicinterneurons restores balance to neuronal circuitry that has beenaffected by neurodegenerative diseases such as PD.

Example 1

Materials And Methods

Experimental Design

6-OHDA lesions were induced on experimental day 1 and performedbehavioral tests on weeks 3 and 5. In rats selected for grafting, MGEcells were transplanted on week 6, and behavioral tests were repeated onweeks 9, 11, 14 and 18 (FIG. 1, Panel A). All animals were treated inaccordance with protocols approved by the Institutional Animal Care andUse Committee at UCSF.

6-OHDA-Model

Unilateral lesions of the nigrostriatal projection in rats, using6-OHDA, leads to the loss of dopaminergic cells in the SNc throughretrograde transport, and loss of dopaminergic terminals in the striatumthrough axonal disruption (Berger et al., 1991). As a consequence, thedistribution of D1 and D2 receptors is altered. Unilateral damage canresult in bilateral changes in the SNc (Berger et al., 1991). Damage ofthe nigrostriatal pathway in rats is accompanied by a compensatoryincrease in the synthesis and release of dopamine from the dopaminegicterminals that remain (Zigmond et al., 1984). To evaluate the success ofsurgery, a subset of animals (n=5) were perfused 4 weeks after lesionand the SNc stained for tyrosine hydroxylase immunoreactivity (TH-IR), alimiting enzyme in the synthesis of dopamine, in order to labeldopaminegic cells. In successful surgeries, the side of the SNcipsilateral to the 6-OHDA injection did not show TH-IR, while thecontralateral side had numerous TH+ cells (FIG. 1, Panel B). To evaluatethe 6-OHDA surgeries in vivo, behavioral testing was performed(described below).

6-OHDA-Surgery

Adult female rats were anesthetized with ketamine (90 mg/Kg) andxylazine (7 mg/Kg), and when insensitive to pain, immobilized within astereotaxic frame in flat skull position. A two-centimeter mid-sagittalskin incision was made on the scalp to expose the skull. The coordinatesfor the nigrostriatal bundle were determined based on the Paxinos andWatson adult rat brain atlas (Paxinos and Watson, 1982). A hole wasdrilled through the skull at the appropriate coordinates, and a glasscapillary micropipette stereotaxically advanced so that the internal tipof the pipette was located within the nigro-striatal pathway. Themicropipette had a 50 μm diameter tip and was filled with a solution of6-OHDA, 12 gr/3 μl in 0.1% ascorbic acid-saline. The 6-OHDA was injectedinto the right nigro-striatal pathway at a rate of 1 μl/minute. Themicropipette was kept at the site for an additional 4 minutes beforebeing slowly withdrawn. The skin incision was closed with stainlesssteel wound clips. Each animal was injected with 6-OHDA on the rightside only, producing hemi-Parkinsonian rats.

MGE Transplant Surgery

Adult pregnant rats that carried the green fluorescent protein (EGFP)under the chick-beta actin promoter (Wistar-TgN (CAG-GFP, 184Ys, RatResource and Research Center) were anesthetized as described above.Fetuses were removed from the uterus at E14.5 and brains dissected undera microscope. The MGE was dissected from the forebrain in oxygenatedartificial cerebrospinal fluid (aCSF, in mm: NaCl, 125; KCl, 2.5; MgCl2,1; CaCl2, 2; NaPO4, 1.25; NaHCO3, 25; and glucose, 25; Sigma). EightMGEs were used to prepare dissociated cells for each surgery. The MGEtissue was mechanically dissociated in the presence of 1% DNAse I inaCSF. Cells were centrifuged at 2× gravity for 2 minutes, and pelletsdissociated in 5 μl of aCSF. Cells were injected immediately afterdissociation. Three injections were performed along the rostro-caudalaxis of the striatum, and cells were deposited at three delivery sitesalong the dorsal-ventral axis at each injection site, starting with themost ventral site first and then withdrawing the injection pipettedorsally to perform the second and third injections. 400 nl of cellsuspension was injected at each delivery site, and a total of 3.6 μl ofMGE cell suspension was injected in each striatum. One 300 nl injectionwas perfomed into the STN. The total number of transplanted cells were252,390±7729 (n=3 rats).

Immunocytochemistry

Rats were anesthetized as described above and perfused intracardiallywith 0.1M phosphate buffer saline (PBS) followed by 4% paraformaldehydein PBS. The brains were removed and post-fixed 24 hours in the sameparaformaldehyde at 4° C. Coronal 50 μm slices were prepared on avibratome (Leica). Free-floating sections were blocked in 10% donkeyserum (Gibco, CA, USA), 0.1% triton X-100 (Sigma, Mo., USA) and 0.2%gelatin (Sigma, Mo., USA). Sections were incubated 24 hours in primaryantibodies at room temperature. The primary antibodies were:anti-neuronal specific nuclear protein (mouse anti-NeuN, Chemicon),Gamma Aminobutryic Acid (rabbit anti-GABA (1:1000, Sigma), Glutamic AcidDescarboxylase 67 (mouse anti-GAD, 1:1000, Abcam), GABA Transporter 1(rabbit anti-GAT, 1:300, Abcam), CNPase (mouse anti-1:500, Abcam), GlialFibrillary Acidic Protein (rabbit anti-GFAP, 1:1000; Sigma), Substance P(rabbit anti-SP, 1:2000, Chemicon), Somatostatin (mouse anti-Som, 1:100,Abcam), Nitric Oxide Synthase (rabbit anti-NOS, 1:100, Abcam), CholineAcetyltransferase (goat anti-Chat, 1:100; Chemicon), TyrosineHydroxylase (mouse anti-TH, 1:1000, Boehringer Mannheim Biochemica),Parvalbumin (mouse anti-PV, 1:1000, Chemicon), Calretinin (mouseanti-CR, 1:1000; Chemicon), Calbindin (mouse anti-CB, 1:2000; Swant,Bellinzona, Switzerland), Synaptophysin (mouse anti-Synaptophysin,1:200, Sigma), Dopamine- and cAMP-regulated Protein (rabbit anti-DARPP32, 1:50, Abcam), Dopamine Transporter (rat anti-DAT, 1:500, Abcam),Vesicular Monoamine Transporter 2 (rabbit anti-VMAT, 1:1000, Abcam),Dopamine Receptor 1 (rabbit anti-DR1, 1:2000, Abcam), Dopamine Receptor2 (rabbit anti-DR2, 1:500, Abcam), and green fluorescent protein(chicken anti-GFP, 1:1000, Abcam). Sections were rinsed and incubated inthe appropriate secondary antibody: Cy2, Cy3 or Cy5 conjugatedpolyclonal anti-mouse/goat/rabbit antibodies (1:100, JacksonLaboratories, ME, USA). Tissue was rinsed and mounted on coated glassslides and cover-slipped with an aqueous mounting medium (Aquamount;Lerner, Pa., USA). Confocal microscopy was performed on an OlympusFluoview confocal laser scanning microscope and analysis performed inFluoview v.3.3 (Olympus). The first antibody was omitted as a controlfor each immunostaining experiment.

Cell Quantification

The survival of MGE cells after transplantation, and the percentage oftransplanted MGE cells that expressed cell specific markers werequantified through confocal microscopy. Brains were perfused asdescribed above at three days, one week, two weeks, three weeks, fourweeks, eight weeks and 12 weeks after transplantation. Brains were cutinto 50 thick coronal sections as described above. The number of MGEcells that survived transplantation was estimated by quantifying thenumber of cells in every third 50 μm thick section throughout therostro-caudal axes of the brain, and multiplying this number by three.The percentage of MGE transplanted cells that expressed cell-typespecific markers was calculated using at least three animals for eachmarker (n=27 total animals analyzed). MGE cells were counted in threecoronal sections: one section at the level of a MGE celltransplantation, a second section 300 μm rostral to the injection, and athird section 300 μm caudal to the site of injection. The total numberof GFP+MGE cells in each section were counted and the percent that werepositive for NeuN, GABA, GAD, GAT, CR, CB, PV, Som, Substance P, NOS,Chat, CNPase, and synaptophysin were quantified at three time pointsafter transplantation (four, eight and 12 weeks).

Electrophysiology

Coronal slices for electrophysiological recordings of GFP+MGEtransplanted cells were prepared as described previously (Noctor et al.,2008). Briefly, brains were sectioned at 400 μm with a vibratome (Leica)in ice-chilled artificial cerebrospinal fluid (aCSF) bubbledcontinuously with 95/5% O2/CO2. containing (in mM): NaCl 125, KCl 5,NaH2PO4 1.25, MgSO4 1, CaCl2 2, NaHCO3 25, and glucose 20, pH 7.4, at25° C., 310 mOsm/l. Tissue slices were allowed to rest for one hourbefore recording. Slices were transferred to a recording chamber on anOlympus BX50WI upright microscope and were perfused with aerated aCSF.GFP+ cells were identified under epifluorescence and recordingsperformed using an EPC-9 patch-clamp amplifier (Heka Electronics)controlled by an Apple computer running Pulse v8.0 (Heka). Glassrecording electrodes (7-10 MΩ) were filled with (in mM): KCl 130, NaCl5, CaCl2 0.4, MgCl2 1, HEPES 10, pH 7.3, EGTA 1.1, to which 500 μM Alexa594-conjugated biocytin (Molecular Probes) was added to identifyrecorded cells. Epifluorescent images of the recorded cells werecollected using Scion Image, and arranged using Photoshop.Electrophysiological responses were measured and analyzed using Pulse,and traces were arranged using Igor Pro (Wavemetrics), and Freehand(Macromedia).

Behavioral Tests

Behavioral tests were performed 3 and 5 weeks after 6-OHDA surgery and3, 5, 8 and 12 weeks after MGE cell transplants. All animals were caredfor according to protocols approved by the Institutional Animal Care andUse Committee at UCSF.

Rotational behavior: Each rat was injected with the dopamine agonistapomorphine (0.05 mg/kg, IP) to produce contralateral rotationalbehavior in 6-OHDA treated rats. Drug-induced rotations were measured inan automated rotometer bowl 28 cm in diameter×36 cm high (ColumbusInstruments, Ohio, Brain Research, 1970, 24:485-493). Afterintraperitoneal injection of apomorphine, the animals were fitted with ajacket that was attached via a cable to a rotation sensor. The animalswere placed in the test bowl and the number and direction of rotationswas recorded over a test period of 40 minutes. This test wasadministered to each rat to verify and quantify the efficacy of theintracranial 6-OHDA-infusion (FIG. 1, Panel C). Apomorphine stimulatesdopaminergic receptors directly, preferentially on the denervated sidedue to denervation induced dopamine receptor supersensitivity, causingcontralateral rotation (Creese et al., 1977; Ungerstedt, 1971;Ungerstedt and Arbuthnott, 1970). There is a threshold of SNc damagethat must be reached in order to produce maximal rotation behavior afterapomorphine administration (Hudson et al., 1993). The abnormal behaviorof hemi-Parkinsonian rats is directly related to the amount of DA cellloss. When there is less than 50% dopamine depletion in the striatumasignificant change in rotation behavior after apomorphine injection wasnot observed, due to compensatory mechanisms in the striatum. For thegrafting experiment only those 6-OHDA rats that rotated at least fourtimes more to the contralateral than to the ipsilateral side of theinjection were selected.

Stride Test:

The test animal was placed on a runway 1 m long and 33 cm wide withwalls 50 cm high on either side. The runway was open on the top, and wassituated in a well-lit room. A dark enclosure was placed at one end ofthe runway, and rats were free to enter the enclosure after traversingthe runway. Rats were trained to run down the runway by placing them onthe runway at the end opposite to the dark enclosure. The practice runswere repeated until each rat ran the length of the runway immediatelyupon placement in the runway. The floor of the runway was covered withpaper. At the start of each test, the animals' rear feet were dipped inblack ink before being placed at the beginning of the runway. The testtwice was repeated for each rat and the length of stride for each testwas measured to obtain an average stride length for each rat. Theaverage stride length was compared across groups. 6-OHDA rats displayimpairments in the posture and movement of the contralateral limbs. Theycompensate by supporting themselves mainly on their ipsilateral limbs,using the contralateral limb and tail for balance, and bydisproportional reliance on their good limbs to walk. The good limbs areresponsible for both postural adjustments and forward movements and theyshift the body forward and laterally (Miklyaeva, 1995). The bad limbproduces little forward movement, and as a consequence the length ofstep is shorter in 6-OHDA rats than in control rats (FIG. 1, PanelsD,E).

Width of the Path Test:

Normal control rats ran straight down the runway to the enclosure at theend. In 6-OHDA rats, however, the limb impairment produced a wanderingpath that zig-zagged from side to side, and as a consequence the pathwayfollowed by the 6-OHDA rats was wider than normal. The maximum width ofthe path for control and experimental groups were compared (FIG. 1,Panel F).

Open Field Test:

An Open Field 16×16 Photobeam System with Flex-Field software (SDInstruments) was used to record the complete coordinates of a rat'smovements within an acrylic enclosure (40 cm wide, 40 cm deep and 37.5cm high). Each animal was placed in the center of the enclosure at thestart of the test and allowed to freely explore the apparatus for 5minutes. The movement of the rat interrupts the laser photobeams. TheFlex-Field software recorded units of movement, which were representedby the number of entries into each of the 256 square zones of the openfield, and the amount of time spent at each point. At the end of theexperiment the movement of each rat during the 5 minutes period wasrepresented as units of movement, and by a zone map that traced the pathfollowed by the rat in the open field.

Results

MGE Cells Survived Up to One Year after Transplant into the AdultStriatum

To determine if MGE cells survive in the adult striatum, 6-OHDA ratstransplanted into the striatum with MGE cells carrying the GFP reportergene were sacrificed at various times after transplant. Of theapproximately 250,000 cells injected, most died during the first 3 weeksafter transplant. By week 4, approximately 1% (2,613±156) of thetransplanted cells were present. These cells, however, persisted, andthe number of surviving transplanted cells did not decrease further at 8and 12 weeks and for up to one year after transplantation. To determineif the transplanted MGE cells continue to proliferate aftertransplantation the fixed tissue was labeled with Ki67, a markerexpressed by dividing cells. Ki67+ MGE cells were not found at four,eight and 12 weeks after transplantation, indicating that even if theMGE cells were initially proliferative, they had ceased dividing withinfour weeks after transplantation.

Transplanted MGE Cells Migrate Throughout the Striatum

The GFP reporter gene expression in the MGE transplant cells allowed forthe examination of the morphology and behavior of the surviving MGEcells. At 3 and 7 days post-transplant, GFP+ cells had the classicalmorphology of migrating neurons, including a simple bipolar shape and aleading process. At two weeks post transplant many of the transplantedcells had migrated up to 2.0 to 2.5 mm in all the directions from thesite of transplantation, and some of the transplanted cells had migratedup to 3.5 mm. At two weeks post transplant the cells appeared moremature and displayed abundant neuritic processes. The vast majority, ifnot all GFP+ cells migrated within the striatum and did not migrateoutside its borders (FIG. 2, Panel A).

After 4 weeks, the transplanted MGE cells had a fully mature appearance.Most of them had an oblong cell body and numerous processes thatextended at least 50 □m. Each process extending from the transplantedcell gave rise to second, third and fourth order processes. Eight and 12weeks after transplantation most GFP+ cells were found within 2.5 mm ofthe injection. Grafted cells had very extensive and ramified processesthat could be observed as GFP+ fibers or puncta throughout thetransplanted striatum. Therefore, GFP+ transplant cell processes appearto occupy the entire striatum, including areas remote from the injectionsites. (FIG. 2, Panel B).

Most Transplanted MGE Cells Transformed into Inhibitory GABA+ Cells

The fate of the transplanted MGE cells was examined by quantifying thepercentage of MGE cells that expressed cell-specific markers at four,eight, and 12 weeks after transplant. At four weeks aftertransplantation, the majority of the transplanted MGE cells haddifferentiated into neurons since 75% expressed the mature neuronalmarker NeuN+(75±6%, n=676). Most of the transplanted cells alsoexpressed markers of GABAergic neurons, including GABA (75±4%, n=294),the GABA synthesizing enzyme GAD (60±11%, n=382), and the GABAtransporter GAT (50±9%, n=380, see FIG. 3). These data indicate that themajority of MGE transplanted cells became GABAergic neurons.

Next, the transplanted cells were examined for the expression ofadditional markers that are typical of striatal neurons in the adultbrain. Some of the GFP+ cells expressed markers of striatal interneuronsubtypes such as the calcium binding protein calretinin (CR, 8.3±0.5%,n=473) and parvalbumin (PV, 0.5±0.5%, n=320), Som (1±0.5%, n=300), orNOS (1±0.7%, n=337). A small percentage of the transplanted MGE cellsexpressed the cholinergic interneuron marker Chat, which is expressed bystriatal excitatory interneurons. Some of the transplanted GFP+MGE cellsexpressed markers that are typical of striatal inhibitory projectionneurons such as CB (24±7%, n=300) and Substance P (6±2%, n=193) (forreview (Tepper and Bolam, 2004) (FIG. 3).

Transplanted cells were also examined for expression of markers that arerelated with dopamine synthesis, but none of the observe transplantedcells expressed VMAT or DAT. Additionally, DARPP 32 expression, a markerof adult medium spiny projection neurons, was not expressed by MGEcells. Together these data indicate that most MGE cells transplantedinto adult striatum differentiate into local GABAergic interneurons andexpress a range of cell specific markers that are normally expressed inthe adult rat striatum.

The pattern of marker expression of grafted cells was largely preservedat 8 and 12 weeks, except for the number of Som expressing cells thatrose slightly at 12 weeks after transplantation (10±3%, n=343). Incontrast, by 12 weeks after transplantation, the number of MGE cellsthat expressed the calcium binding proteins was reduced (CR, 2±1%,n=258; CB, 2±1.5% n=270; PV, 0%, n=136). Interestingly, thetransplantation of the MGE cells temporarily induced strong expressionof CR and CB by host cells surrounding the injection site. The strongexpression at four weeks after transplantation, was not present at 8 or12 weeks. Host cells in the control animals that received only vehicledid not express CR or CB, indicating that the expression was likely dueto presence of the grafted cells.

Approximately, one fourth of the grafted MGE cells (25±4%) wereNeuN-negative. These cells stained positive for the myelin protein,CNPase, indicating that a subpopulation of the grafted cellsdifferentiated into oligodendrocytes. None of the cells transplantedinto the striatum expressed GFAP suggesting that grafted cells did notdifferentiate into astrocytes. The ratio of neuronal to glial cells(3:1) was maintained at 8 and 12 weeks post transplantation.

MGE Transplanted Cells Integrated into Striatal Circuitry

The MGE transplanted cells were examined for the establishment ofsynaptic connections in the striatum and functionally integratation. Atfour weeks after transplantation, 67±3% of the GFP+MGE cells (n=193)expressed synaptophysin puncta along their processes, indicating thepresence of synapses (FIG. 4, Panel A). Electrophysiological recordingsprovided further evidence that the MGE cells became functionallyintegrated into the host striatum. Whole-cell patch-clamp recordingswere obtained from GFP+MGE cells 20 weeks after transplantation toexamine their basic membrane properties (n=13 cells). Alexa-594 dye wasincluded in the patch electrodes to confirm that recordings wereobtained from targeted GFP-expressing transplant cells (FIG. 4, PanelB). The recordings demonstrated that most of the transplanted MGE cellsbecame functionally mature neurons (11/13 cells). The neuronal cells hada resting membrane potential of 65.9±5.1 mV. In voltage clamprecordings, a series of depolarizing voltage steps elicited voltagegated inward currents characteristic of neurons (FIG. 4, Panel C). Incurrent clamp recordings, a series of depolarizing current stepselicited mature action potentials (APs) with a spike amplitude of69.1±4.6 mV, and large after-hyperpolarizations of 14.5±2.9 mV (FIG. 4,Panel D). In addition, the transplanted MGE cells fired repetitivenon-accommodating APs, (FIG. 4, Panel D) and trains of spontaneous APs(FIG. 4, Panel F). The membrane properties of the transplanted cellswere consistent with that of MGE derived interneurons in the cerebralcortex (Alvarez-Dolado et al., 2006; Butt et al., 2005), and with themembrane properties recorded from host striatal interneurons (Kawaguchi,1993).

Indeed, some of the transplanted MGE cells fired APs at frequenciesgreater than 100 Hz, indicating a fast-spiking interneuron phenotype.The transplanted MGE cells also displayed evidence that they werereceiving synaptic inputs. In voltage clamp recordings the GFP+ neuronsexhibited spontaneous synaptic currents at a frequency of 1.1, 0.2 Hz(FIG. 4, Panel G), which was similar to the frequency observed inrecordings obtained from control host striatal cells (1.0±0.3 Hz, n=5host cells). These results are consistent with those of Alvarez-Doladoet al. (Alvarez-Dolado et al., 2006), who showed that transplanted MGEcells increased the amount of GABAergic inhibition on host neocorticalprojection neurons. Two of the recorded GFP+ cells did not expressvoltage-dependent inward currents, and did not fire action potentialswhen stimulated with a series of depolarizing currents (FIG. 4, PanelE). These recordings were most likely obtained from the non-neuronal,CNPase+ population of transplanted cells, and are consistent with themembrane properties of mature glial cells in the cortex. Together thesedata support the conclusion that most transplanted MGE cells becomeinhibitory interneurons and are synaptically integrated into the hoststriatal circuitry.

MGE Cell Striatal Grafts Ameliorated the Behavioral Symptoms of 6-OHDALesioned Rats

The behavioral effect of MGE cells on 6-OHDA lesioned rats was examinedusing three behavioral tests performed before and after transplantation(FIG. 5).

Rotation Under Apomorphine

As previously shown, upon apomorphine administration, unilaterally6-OHDA lesioned rats rotated significantly more to the contralateralside (with respect to the lesioned side) than the ipsilateral sidecompared to control rats that rotated approximately equally in bothdirections. After transplantation, there was a significant reduction inthe number of contra-lateral turns in the MGE transplanted 6-OHDAlesioned rats (n=21) compared to non-transplanted 6-OHDA controls(n=12). This effect was observed at all experimental times beginningwith week 9 (p<0.05) and continued until the last time point, 18 weeks(p<0.05). The performance of sham-transplanted 6-OHDA rats (n=11) wasundistinguishable from non-transplanted 6-OHDA rats (n=12), indicatingthat the MGE cells, and not the transplantation procedure, wereresponsible for the motor improvement of MGE-transplanted 6-OHDA rats.(FIG. 5, Panel a).

Apomorphine binds to dopamine receptors expressed by host striatalneurons, which causes rotation in the 6-OHDA rat (Ungerstedt andArbuthnott, 1970). Apomorphine-induced rotational behavior wassignificantly reduced after MGE transplantation suggesting that thetransplanted MGE cells express dopamine receptors and are directlyresponsible for the observed reduction in rotational behavior. Thishypothesis was confirmed by performing fluorescence immunostaining fordopamine receptor 1 (DR1) and 2 (DR2). Both were detected in somaticmembrane and processes of MGE cells 12 weeks after transplantation (FIG.6). These data demonstrate that apomorphine directly stimulates MGEinhibitory interneurons, altering the balance of excitation/inhibitionin the striatum and modifying rotational behavior.

Length of Stride

6-OHDA rats (n=12) had a stride length of 13.2±0.2 cm, significantlyshorter than the stride length of control rats, 15.1±0.3 cm (p<0.001,n=16). After MGE cell transplantation, the stride length of the 6-OHDArats (n=21) increased and by week 9 reached values similar to those ofcontrol rats (15.5±0.4 cm, n=16). The increase in the length of stridewas maintained after 11 and 14 weeks. The stride length of 6-OHDA ratsthat received a sham transplant (n=11) did not change, and was notsignificantly different from that of 6-OHDA rats that received notreatment (n=12) (FIG. 5, Panel b).

Width of Path

The maximum path width of all rats was measured as they ran down therunway (FIG. 1, Panel e). 6-OHDA animals (n=12) had a path width thatwas 3.4±0.3 cm, which was significantly wider than the value of 2.5±0.3cm for control rats (p<0.05, n=16). The path width of 6-OHDA rats thatreceived MGE cell transplants decreased and by week 11 matched (2.7±0.3cm, n=21) that of the unlesioned control animals (n=16). The path ofsham transplanted 6-OHDA rats (n=11) was not significantly differentfrom that of 6-OHDA rats (n=12). These data indicate that MGEtransplantation resulted in a substantial improvement in the gait of6-OHDA lesioned rats (FIG. 5, Panel c).

MGE Cell Striatal Transplants Alter the Motor Performance of Wild-TypeRats

MGE cells were grafted into intact rats to examine the effect of MGEcells on unlesioned animals using the behavior assays described above.The path width and the rotations under apomorphine were notsignificantly different in rats injected with MGE cells compared tonon-transplanted controls. Interestingly, the stride length of wild-typerats that received MGE grafts was significantly longer than that ofcontrol untreated rats (p<0.001, n=6) (FIG. 7, Panel a). This wasbelieved due to an increase in motor activity after MGE-transplantation.To confirm this hypothesis an open field activity test was performed onwild-type control, 6-OHDA injected and MGE-transplanted wild-type ratsusing a five minute test period (FIG. 7, Panel b). Surprisingly,MGE-cells transplanted into control rats (no 6-OHDA treatment) produceda significantly higher level of motor activity (1101±39 UM) than inuntreated control rats (912±104 UM). 6-OHDA injected rats, as expected,had lower motor activity levels than control rats (453±69 UM). Theincrease of motor activity in wild-type rats versus control rats isreflected in the open field zone map (FIG. 7, Panel c).

MGE Cells Transplanted in the Subthalamic Nucleus Survived, but Did notMigrate, and Transformed into Glial Cells

The results observed after MGE cell transplantation into the striatumsuggests that increased GABAergic inhibition in the striatumsignificantly ameliorates motor symptoms in hemi-Parkinsonian rats. Ithas been suggested that increasing inhibition in the subthalamic nucleus(STN) could also have beneficial effects in Parkinson's disease (Benabidet al., 2000; During et al., 2001). Therefore, the survival anddifferentiation of grafted MGE cells were tested in this nucleus. Thebehavior and phenotype of MGE cells, however, was strikingly differentafter transplant into the STN. In contrast to MGE cells that migratedsubstantial distances after transplant into the striatum, MGE cellstransplanted into the STN did not migrate from the site of injection(n=6, FIG. 8, Panel A). Moreover, the MGE cells in the STN did notbecome neurons. None of the MGE cells in the STN expressed the neuronalmarker NeuN (n=231). In contrast, a small number of MGE cells locatedoutside the boundary of the STN did express NeuN (FIG. 8, Panel B).Furthermore, none of the grafted MGE cells in the STN expressed thecalcium sequestering proteins CR or CB, or the inhibitory interneuronmarkers GAD or GABA (FIG. 8, Panels E, G, H, I). While none of graftedMGE cells in the striatum expressed the astrocyte marker GFAP, themajority of MGE cells in the STN expressed GFAP (75±5%, n=151). Roughlyone third of MGE cells also expressed the oligodendrocyte marker CNPase(30±9%, n=132, FIG. 8, Panels C,D,J,K) A small percentage of MGE cellsin the STN expressed the GABA transporter GAT1 (13±5%, n=94, FIG. 8,Panel F). The GAT expressing cells were most likely astrocytes oroligodendrocytes which have been shown to express the GAT1 GABAtransporter (Pow et al., 2005). These data indicate that, in contrast tothe striatum where MGE grafted cells integrate and differentiate intoneurons, most grafted MGE cells in STN differentiate into glial cells.

Example 2

In this study, MGE cell transplantation was tested as a means to addinhibitory GABAergic interneurons to the striatum. Additionally, MGEcell transplantation was tested to see if it would beneficially improvethe motor behavior of hemi-Parkinsonian rats. These studies utilized MGEcells from E14.5 GFP rats that were transplanted into the striatum ofadult rats. A small population of these transplanted cells, migrated,survived and differentiated into GABAergic neurons that becamesynaptically integrated into the striatal circuitry. MGE cells that weregrafted into the striatum of 6-OHDA lesioned rats alleviated motorsymptoms of PD. Fetal MGE grafts in the striatum also impacted thebehavior of normal untreated rats; the transplanted MGE cells increasedseveral measures of motor activity in the control animals. The abilityof MGE cells to differentiate into interneurons appears to be dependenton the host environment since transplanted MGE cells into the STNdifferentiated into astrocytes or oligodendrocytes rather than neurons.These data indicate that injection of MGE cells into the striatum is anew modality to modulate hyperactive neurons in the striatum andtherefore alleviate motor symptoms in PD.

MGE Cells Transplanted into the Adult Striatum Transform into InhibitoryInterneurons and Integrate into the Striatal Circuit

MGE cells transplanted into the striatum survived at least one year,migrated from the site of injection, and were widely distributedthroughout the striatum. This study demonstrated that MGE cellstransplanted into the adult striatum acquire a mature morphologicalphenotype and differentiate into GABAergic neurons that express markerscommon to neostriatal interneurons. Although a small percentage of theMGE transplanted cells survived after 3 months, the ability of thesecells to impact behavior depends on their number in relation to thenumber of host interneurons. Stereological based studies estimate thatthe number of striatal interneurons in the adult rat is approximately49,000: 13,000 CR+ interneurons (Rymar et al., 2004), 15,000 PV+interneurons (Larsson et al., 2001; Luk and Sadikot, 2001) and 21,000SOM+ interneurons (West et al., 1996). Therefore, the average number oftransplanted MGE cells that survived in the current experiments(2613±156, n=3) represents approximately 5% of the total number ofnative striatal interneurons. In addition, the MGE cells extendednumerous processes throughout the striatum, indicating their potentialfor interacting widely with host cells. The number of surviving MGEcells are sufficient to produce motor behavioral changes, both in 6-OHDAtreated, and untreated control animals. MGE cells unexpecteddemonstrated the generally effect of increasing motor speed of alltreated animals, including the unlesioned, control animals. Thetransplantation of a higher number of MGE cells is anticipated to resultin a greater number of surviving interneurons in the host striatum.

The embryonic MGE produces both striatal and neocortical interneurons,and both populations share the expression of many of the same sets ofmarkers (Defelipe et al., 1999; Kawaguchi, 1997; Kubota et al., 1994).Thus, it is likely that the dissociated embryonic MGE cells that wereinjected into the striatum included a mixture of cells destined topopulate both the striatum and the neocortex, and these cells retainedtheir normal molecular expression characteristics after transplantation.However, the pattern of cell markers expressed by MGE cells wasinfluenced by their post transplantation environment as most MGE cellsthat survived in the adult STN nucleus expressed GFAP and none expressedneuronal markers.

GFP+ processes were not detected outside of the striatum in thetransplanted animals, indicating that the transplanted MGE cells did notproject outside of the striatum as do GABAergic projection neurons.Thus, the MGE cells integrated in the circuitry of the striatum and thebehavioral changes observed in transplanted rats were due tomodification of activity of synapses and/or neurons within the striatumby the transplanted cells. This integration modulated the activity levelof host GABAergic interneurons and/or projection neurons and produced aremarkable improvement in the behavioral deficits.

Transplanted MGE Cells Ameliorate Behavioral and Movement Deficits in6-OHDA Rats

6-OHDA lesioned rats that received MGE cell grafts exhibited behavioralimprovements including improvement in the apomorphine rotational test,an increase on the length of stride, and a normalized gait. Thesebehavioral and movement changes indicate a general improvement of themotor symptoms of PD animals after MGE transplantation.

MGE cells were transplanted six weeks after the induction of 6-OHDAlesions. The MGE cells differentiated into GABA+ interneurons within 4weeks after transplant and notably improved motor symptoms of PD. Theseresults demonstrate that, in addition to the protective effect describedpreviously, GABA also improve PD symptoms once the disease isestablished. The ability of MGE cells to disperse in the striatum,mature, remain functionally active, and synaptically integrate intobasal ganglia circuitry is responsible for the observed behavioralmodifications. MGE cells can also carry trophic factors orneurotransmitter synthesizing enzymes to further ameliorate symptomsproduced by dysfunctional circuits in the striatum. Useful neurotrophicfactors include the neurotrophins (e.g., NGF; brain-derived neurotrophicfactor, BDNF; and neurotrophins NT-3 and NT-4/5); the neuropoieticcytokines (e.g., ciliary neurotrophic factor, CNTF); and the fibroblastgrowth factors (e.g., acidic and basic FGF). Useful neurotransmittersinclude acetylcholine, epinephrine, norepinephrine, dopamine, serotonin,melatonin, glutamic acid, gamma aminobutyric acid (GABA), aspartic acid,and glycine.

The increase in motor behavior observed after transplantation of fetalMGE cells into the striatum of control rats, indicates that MGE cellsare ideal for achieving a change in the balance of excitatory andinhibitory signals in the striatum, and thus represent a powerfulstrategy for altering output of the striatum in disease conditions.

Therapeutic Advantages

The approach of the methods of the present disclosure is to injectdissociated cells from the embryonic MGE into the adult basal ganglia ofa subject having or suspected of having PD, epilepsy or otherneurological condition amenable to treatment according to the methodsdisclosed herein. The advantage of MGE cell transplants over other celltypes is their capacity for dispersal throughout the striatum. Forexample, mesencephalic cells show very little migration aftertransplantation into the striatum and remain in clumps within the basalganglia. These clusters are thought to be responsible for some of theserious side effects that result from transplantation of dopaminergicprecursors for PD (Bhattacharya et al., 2004). In contrast, the MGEcells have the surprising ability to disperse widely aftertransplantation and integrate into the host striatum in a homogenousmanner that lessens PD symptoms without undesired side effects seen inother therapies. MGE grafts do not produce tumors or cause aberranttissue organization. Striatum grafted MGE cells increase motor activitylevels, demonstrating that MGE cells change the balance of excitatoryand inhibitory activity. In some embodiments, MGE grafts reduce one ormore TRAP features of PD and/or reduce flexed posture and freezing by atleast 10% compared to controls or the previous untreated state. In someembodiments, MGE grafts reduce one or more TRAP features of PD and/orreduce flexed posture and freezing by at least 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 95% compared to controls or the previous untreatedstate. In some embodiments, MGE grafts reduce the amount of requireddrug by at least 10% compared to controls or the previously untreatedstate. In some embodiments, MGE grafts reduce the amount of requireddrug by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% comparedto controls or the previously untreated state. In some embodiments, MGEcells transplanted into the striatum survive for at least 6 months, Insome embodiments, MGE cells transplanted into the striatum survive forat least 1, 2, 3, 4, 5, 7, or 10 years. In some embodiments, treatmentof patients suffering from bradykinesia is preferred. In someembodiments, before and after treatment success is measure under atleast one of the following criteria, the unified Parkinson's diseaserating scale (UPDRS), motor “off” (off medication) scores, OFF time anddyskinesias, and dose of L-Dopa.

Example 3 Epilepsy

Transplanted MGE cells, harvested from mice expressing green fluorescentprotein (GFP), co-label with antibodies to GABA, GAD67, calretinin,parvalbumin, neuropeptide Y or somatostatin at 30 days aftertransplantation (30 DAT) (FIG. 9, Panels A, B). In neocortical tissueslices from 30 DAT grafted animals, MGE-GFP neurons primarily exhibitfiring properties similar to endogenous interneuron sub-types: (i)fast-spiking, (ii) regular-spiking non-pyramidal cells and (iii)burst-spiking non-pyramidal (FIG. 9, Panels C-D). Consistent withprevious observations of graft-derived cells with immature and“migratory” anatomical profiles immediately following transplantation(Wichterle 1999; Alvarez-Dolado 2006), current-clamp recordings at 7 to10 DAT revealed GFP+ neurons with immature intrinsic membrane propertiese.g., small and broad width action potentials, depolarizing restingmembrane potential and high input resistance (data not shown). Asexpected at 30 DAT (Alvarez-Dolado 2006), GABA-mediated input topyramidal neurons in the host brain, measured as inhibitory postsynapticcurrent (IPSC) frequency, increased by nearly 300% in mice receiving MGEgrafts (control: 0.6±0.1 Hz, n=8; MGE: 2.3±0.3 Hz, n=7).

MGE progenitors were examined for their ability to generate interneuronsthat would innervate native interneurons and alter the activity of theendogenous interneurons, an undesirable side effect. To determinewhether GABA-mediated inhibition onto endogenous interneurons wasaltered, the spontaneous IPSCs in the host neocortex of grafted micewere examined. Interneurons were identified by their oval, oftenmulti-polar morphologies under IR-DIC (FIG. 10, Panel a), confirmed posthoc with biocytin labeling (FIG. 10, Panel b) and voltage-clamped inbath solution containing glutamate receptor antagonists (DNQX and APV;FIG. 10, Panel C) to isolate GABA-mediated current. IPSC analysis failedto identify significant differences between control (un-transplanted)and grafted mice at 30 DAT (FIG. 10, Panels d-f).

Enhanced inhibition in grafted animals could also lead to homeostaticchanges in postsynaptic GABAA receptors (Mody 2005; Xu 2007). Analysisof evoked IPSC kinetics, decay time constants in particular (Soltesz; MVJones), would reflect potential changes in postsynaptic GABA receptorsubunit expression and were examined in neocortical slices (30 DAT).White matter stimulation elicited IPSCs on Layer ii/III pyramidalneurons in regions containing GFP+ cells with kinetic properties thatwere not different between control and grafted animals (FIG. 11, PanelsA and B). Together, these data further support the MGE transplantationprotocol as an effective strategy to selectively enhance inhibition ofprincipal neurons in postnatal brain.

Because some available antiepileptic drugs (AEDs) enhance GABA-mediatedsynaptic transmission and enhanced inhibition could be therapeutic(MacDonald 1989), MGE transplants were examined for their ability toreduce acute seizures. Early postnatal bilateral transplantation ofMGE-GFP progenitors generated new GFP-immunoreactive (IR) cells in hostneocortex with distributions between 0.75 and 5 mm from the injectionsite (FIG. 12, Panels a1-4; also see Alvarez-Dolado 2006). Newinterneuron density was confirmed for all animals used in acute seizurestudies and a threshold of approximately 40,000 GFP-IR cells per animal,with migration ∃0.5 mm from the injection site, was defined as asuccessful graft (FIG. 12, Panel a5). Pilocarpine, a muscarinicacetylcholine receptor antagonist and common convulsant agent, wasadministered to adult wild-type mice at a concentration (300 mg/kg,i.p.) chosen to maximize the number of animals that would experiencetonic-clonic seizures (Winawer et al. 2007). High-amplitude synchronouselectrographic seizure activity was confirmed using scalpelectroencephalographic (EEG) recording and dual digital cameras (FIG.12, Panel B). Behavioral seizure manifestations including forelimbclonus (Stage III, S3; Racine 1972; Jones 2002) were observed in −75% ofcontrol mice at this pilocarpine concentration; the latency to firstbehavioral seizure was between 10 and 15 minutes post-injection, asreported previously (Hamani 2004). In mice grafted with MGE cells at P2,the percentage of animals exhibiting S3 seizures was reduced toapproximately 50% (FIG. 12, Panel c1); latency to first seizure behaviorwas unchanged (FIG. 12, Panel c2). This level of “seizure protection”was comparable to that observed with phenobarbital (20 mg/kg) orcarbamazepine (50 mg/kg) pre-treatment, and better than phenyloin (200mg/kg), an AED previously shown to exacerbate pilocarpine-inducedseizures (Sofia et al., 2003) (FIG. 12, Panel d2).

MGE transplants were then tested for their ability to reduce seizures ina mouse epilepsy model. Spontaneous tonic-clonic seizures were reportedin humans with a dominant-negative missense mutation in KCNA1 (Zuberi1999) or mice with a recessive knockout of Kv1.1/Kcna1 (Smart 1999). Tomonitor spontaneous seizures in these mice, prolongedvideo-electroencephalography (EEG; see Methods for full description ofelectrographic phenotypes) was performed. The EEG of Kv1.1-1-mice showedsevere, generalized electrographic seizures lasting 10-340 s (Grade IV;FIG. 13, Panel A) and occurring more than once per hour (1.5±0.5seizure/hr; FIG. 13, Panel C); electrographic seizures or high voltagespiking were never observed in age-matched wild-type siblings (FIG. 13,Panel A). Video monitoring confirmed tonic-clonic, S4 seizure behavior(e.g., tonic arching, tail extension, followed by forelimb clonus, andthen synchronous forelimb and hindlimb clonus) during ictal seizureepisodes. As reported (Smart 1999; Wenzel 2007; Glasscock 2007),Kv1.1-1-mice exhibit frequent spontaneous seizures starting during thesecond-to-third postnatal week and do not survive beyond the 8thpostnatal week (FIG. 13, Panels a-e); sudden death is likely due tocardio-respiratory failure associated with status epilepticus. Incontrast, Kv1.1-1-mice grafted with MGE progenitors on P2 survive wellpast postnatal week 10 and exhibit a surprising and striking reductionin electrographic seizure activity. Only brief episodes (10-48 s) ofsynchronized high amplitude spiking were observed in transplantedKv1.1-1-mice (FIG. 13, Panels a, b). The frequency of these events wasrare compared to un-transplanted mice (FIG. 13, Panel c) and 43% ofgrafted animals only exhibit interictal spiking (Grade II; Table 1).Kaplan-Maier survival plots show a clear, and statistically significant,rightward shift for Kv1.1 mutant mice receiving successful MGE grafts(FIG. 13, Panel d); new GFP cell density was confirmed as ∃ 40,000 cellsper brain in Kv1.1-1-mice that survived to P57 (FIG. 13, Panels e-f).

Embryonic neural progenitor cells derived from the medial ganglioniceminence generate new interneurons. These cells differentiate intofunctional interneuron sub-types and selectively enhance GABA-mediatedinhibition in the host brain. Newly-generated interneurons demonstrate asurprising protective effect against acute seizures that was comparableto that observed with commercially available AEDs. In some embodiments,MGE progenitor cell transplantation results in a reduction of requiredAED dose of at least 10% compared to untreated controls or to theindividual's previously untreated state. In some embodiments, MGEprogenitor cell transplantation results in a reduction of required AEDdose of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 95%, or 99%,compared to untreated controls or to the individual's previouslyuntreated state. Significant suppression of spontaneous seizures wasobserved in a mouse model of generalized epilepsy. Decreased seizureactivity in these animals was also associated with a delay in suddendeath and significantly prolonged life-span.

Immortalized cells engineered to produce GABA attenuatestimulation-induced electrical after discharge in a rat (Thompson 2000;2005). Attenuation of after discharge duration has also been achievedwith embryonic precursor cells modified to release adenosine (Li 2007)or viral vectors engineered to produce glial-cell line-derivedneurotrophic factor (Kanter-Schlifke 2007), neuropeptide Y (Richichi2004) or galanin (Haberman 2003; McCown 2006). While promising, none ofthese procedures was associated with migration (or transfection) ofcells outside the injection site, functional evidence for modificationof synaptic transmission in the host brain or significant suppression ofspontaneous electrographic seizures. Systemically injected human neuralstem cells modestly reduce behavioral seizures in a rat pilocarpinemodel and hippocampal field recordings suggest an effect on synapticinhibition (Chu 2004). These studies, and the demonstrateddifferentiation of hippocampal neural stem cells into neurons (includingGABAergic interneurons) in a rat model of temporal lobe epilepsy(Shetty, 2005), suggest that interneuron generation and enhancement ofGABAergic neurotransmission (as shown here in single-cell recordings)may be antiepileptic. Although interneuron loss has been reported inepilepsy (de Lanerolle 1989; Spreafico 1998; Golarai 2001), andGABA-enhancing AEDs are used clinically (MacDonald 1986, 1989), aprocedure to modify host brain circuits via the addition of newinterneurons to existing circuits, either normal or pathological, hasnever been demonstrated.

MGE cell grafts were shown to produce a dramatic and remarkablereduction in spontaneous electrographic seizure activity in a mouseShaker-like K+ channel mutant mimicking a human epilepsy phenotype(Kuberi; Noebels). In some embodiments, at least a 10% reduction inspontaneous electrographic seizure activity is achieved. In someembodiments, at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 95%, or 99%reduction in spontaneous electrographic seizure activity is achieved. Insome embodiments, at least a 10% reduction in the duration of seizuresis achieved. In some embodiments, at least a 20%, 30%, 40%, 50%, 60%,70%, 80%, 95%, or 99% reduction in the duration of seizures is achieved.In some embodiments, at least a 10% reduction in the frequency ofspontaneous electrographic seizures is achieved. In some embodiments, atleast a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 95%, or 99% reduction in thefrequency of spontaneous electrographic seizures is achieved. In someembodiments, at least a 10% reduction in high voltage spiking isachieved. In some embodiments, at least a 20%, 30%, 40%, 50%, 60%, 70%,80%, 95%, or 99% reduction in high voltage spiking is achieved. Thisresult is consistent with a long-standing hypothesis that synapticinhibition constrains seizure discharge (Kandel, Dichter, Trevelyan).Moreover, the described transplantation protocol to generate enrichedpopulations of GABAergic interneurons with the unique ability to migrateand functionally integrate in host brain provides a significantadvancement in cell therapy development for the treatment of intractableepilepsy.

Methods

Tissue Dissection and Cell Dissociation.

Ventricular and subventricular layers of the GE germinal region weredissected from E13.5 embryonic GFP+ transgenic mice. The MGE is atransient structure, and at E14 the MGE is well developed but stillseparated from LGE. Bordering tissue between adjacent regions wasdiscarded during dissections to avoid contamination. Explants weremechanically dissociated by repeated pipetting through 200 μl yellowplastic pipette tip (10-20 times). Dissociated cells were washed with 1ml of L-15 medium containing DNase 1 (10-100 μg/ml) and pelleted bycentrifugation (2 minutes, 800 g).

Transplantation.

Highly concentrated cell suspension (˜8×105 cells/μl in 3-5 μl of L-15medium) were front-loaded into beveled glass micropipettes (˜50 μmdiameter) that pre-filled with mineral oil and mounted on amicroinjector. Cells were allowed to settle inside the pipette and theexcess cell-free medium will be expelled. Two-three day old mice wereanesthetized by exposure to −4° C. until pedal reflex was abolished.Anesthesia was maintained by performing surgery on a cold aluminumplate. 5×105 cells/mouse were injected in deep layers of cortex.

Immunostaining.

Animals were transcardially perfused with 4% paraformaldehyde. Brainswere immediately removed, postfixed overnight, and sectioned coronally(50 μm). Floating sections were immunostained with commerciallyavailable antibodies. Secondary antibodies were used: cy3-conjugateddonkey anti-mouse, cy3-conjugated donkey anti-rabbit, cy2-conjugateddonkey anti-rabbit, and biotin-conjugated donkey anti-mouse (all fromJackson ImmunoResearch, PA). Sections were washed in PBS, blocked for 1h in PBS containing 10% donkey serum and 0.1% Triton X-100, thenincubated overnight at −4° C. in primary antibodies diluted in PBScontaining 10% donkey serum and 0.1% Triton X-100, washed 3× in PBS andincubated with secondary antibodies for 1-2 hours at room temperature inthe dark. Fluorescent images were obtained using a cooled-CCD camera(Princeton Instruments) and Metamorph software (Universal Imaging).

Electrophysiology.

Acute neocortical slices were prepared from male or female mice, asdescribed previously (Alvarez-Dolado et al. 2005). Resulting slices wereimmediately transferred to a holding chamber where they remainedsubmerged in oxygenated recording medium (ACSF) consisting of (in mM)124 NaCl, 3 KCl, 1.25 NaH2PO4, 2 MgO4, 26 NaHCO3, 2 CaCl2, 10 dextrose(295-305 mOsm); 37° C. for 45 min and then at room temperature. For eachexperiment an individual slice is gently transferred to a recordingchamber and continuously perfused with oxygenated recording medium atroom temperature. Whole cell voltage-clamp pipette recordings wereobtained from visually identified neurons using an infrared differentialinterference contrast (IR-DIC) video microscopy system. Intracellularpatch solution for whole-cell IPSC current recordings contained (in mM)120 Cs-gluconate, 10 HEPES, 11 EGTA, 11 CsC12, 1 MgC12, 1.25 QX314, 2Na2-ATP, 0.5 Na2-GTP, (pH 7.25; 285-290 mOsm). To isolate GABAergicsynaptic currents, slices were perfused with nACSF containing 20 μM6,7-dinitroquinoxaline-2,3-dione (DNQX) and 50 μMd-(−)-2-amino-5-phosphonovaleric acid (D-APV). IPSCs were recorded atthe reversal potential for glutamatergic currents (holding potential, 0mV; room temperature) where IPSC events exhibit a large amplitude andprominent decay (Otis and Mody 1992). Intracellular patch pipettesolution for current-clamp recordings contained (in mm) 120KMeGluconate, 10 KCI, 1 MgCI2, 0.025 CaCI2, 10 HEPES, 0.2 EGTA, 2Mg-ATP, 0.2 Na-GTP, pH 7.2, (285-290 mOsm). For analysis of intrinsicfiring properties, recordings were obtained from GFP+ cells in theneocortex an IR-DIC microscope equipped with epifluorescence (Olympus).Current and voltage were recorded with an Axopatch 1D amplifier (AxonInstruments) and monitored on an oscilloscope. Cells were depolarizedand hyperpolarized, via direct current injection (5-1000 ms, duration).Whole-cell access resistance was carefully monitored throughout therecording and cells were rejected if values changed by more than 25% (orexceeded 20Ω); only recordings with stable series resistance of <20 MΩwill be used for IPSC analysis (at least 100 events were analyzed percell).

Video-EEG Monitoring.

Behavioral and electroencephalographic (EEG) observations were madeusing a time-locked, dual video digital EEG monitoring system (PinnacleTechnologies, Lawrence, Kans.). For EEG recordings, mice were surgicallyimplanted in the left and right frontoparietal cortex with electrodes.Each mouse was anesthetized with ketamine and xylazine (10 mg/kg and 1mg/kg, i.p. respectively) so that there was no limb withdrawal responseto a noxious foot pinch. Sterile stainless steel recording electrodeswere placed epidurally through burr holes in the skull (one electrode oneither side of the sagittal suture, approximately halfway betweenbregman and lambdoid sutures and ˜1 mm from the midline). Electrodeswere cemented in place with a fast-acting adhesive and dental acrylic,and electrode leads were attached to a microplug that is also cementedto the head of the animal. Animals were allowed to recover for 5 daysbefore experiments are initiated. Experimental animals were monitoredfor between 5-10 days (6-12 hr/day, 5 days/week; at least 30 hr peranimal). A total of 165 hours of recording was analyzed for Kv1.1-1-mice(n=4) and 242 hours for Kv1.1-1-+MGE mice (n=7). Control mice (n=4) wereadministered pilocarpine (300 mg/kg, i.p.) at the conclusion of the lastrecording session to obtain a “baseline” electrographic seizure sample.All monitoring data was coded and scored “blind” for changes in behaviorand EEG by four independent investigators. Electrographic seizures weredefined as follows (modified from Dube 2006; Wong 2007; Henshall 2002):Grade I, basic background, no epileptiform spikes; Grade II, mostlynormal background, some high voltage spikes; Grade III, mostly abnormalbackground with low frequency high voltage spiking and Grade IV, highfrequency, high voltage synchronized poly-spike or paroxysmalsharp-waves with amplitude>2-fold background that last ∃ 6 seconds.Electrographic EEG seizures were analyzed using SireniaScore software(Pinnacle) and confirmed by off-line review of behavioral videorecordings obtained at two different monitoring angles. Behavior wasscored, between Stage 1 (S1) and S4, on a modified Racine scale (Racine;Jones). All animals were sacrificed at the conclusion of EEG monitoringand processed for GFP immunohistochemistry and cell counting.

In some embodiments, patients with temporal lobe epilepsy, the mostcommon form of the disease, are likely candidates for MGE therapy as itis well documented that interneuron loss contributes to the diseasedstate (de Lanerolle et al. 1989). In some embodiments, patients withepilepsy associated with a focal cortical dysplasia are also known tolose interneuron and/or exhibit reduced GABA-mediated inhibition(Spreafico et al. 1998; Calcagnotto et all 2005) and are also goodcandidates for MGE therapy. In some embodiments, clinical neuron-imagingstudies (PET, MRI, etc.) in combination with video-EEG monitoring areused to identify candidate patients. In some embodiments, video-EEGmonitoring and/or seizure self-reporting by patients is used to measureoutcome of the MGE transplantation procedure. In some embodiments, MGEprogenitor therapy success is quantified by a reduction in seizurefrequency or duration in treated patients. In some embodiments, MGEprogenitor therapy success is quantified by a reduction in dosages orthe need for co-treatment with prescribed antiepileptic medications.

Note for figure legend: Kv1.1 is not the dominant Shaker familytranscript expressed in vascular tissue (Cox et al. 2001).Kv1.1-containing potassium channels are predominantly expressed in axonsof the hippocampus and cerebellum (Wang et al. 1993; Rhodes et al. 1997)

TABLE 1 Analysis of EEG data Animal Duration (sec) Frequency (per hr)Highest seizure score Kv1.1 KO #307 38.6 ± 3.7 0.6 ± 0.2 Grade IV #41957.5*16.4 0.8 ± 0.3 Grade IV #461 59.0 ± 9.3 2.5 ± 0.1 Grade IV #46658.0 ± 7.4 2.1 ± 0.6 Grade IV Kv1.1 KO + MGE #428 24.7 ± 2.2 0.1 ± 0.05Grade IV #492 0 0 Grade II #525 0 0 Grade II #533 0 0 Grade II #544 23.7± 4.2 0.2 ± 0.15 Grade IV #573 27.5 ± 4.4 0.2 ± 0.07 Grade IV #575 28.0± 2.1 0.1 ± 0.08 Grade IV

Spasticity

Spasticity is a common disorder in patients with injury of the brain andspinal cord. The prevalence is approximately 65-78% of patients withspinal cord injury (Maynard et al. 1990), and around 35% in strokepatients with persistent hemiplegia (Sommerfeld et al. 2004). Reflexhyperexcitability develops over several months following human spinalcord injuries in segments caudal to the lesion site. Intractablespasticity is also a common source of disability in patients withmultiple sclerosis. Symptoms include hypertonia, clonus, spasms andhyperreflexia.

There are numerous medicines that treat the general effects ofspasticity. These drugs act on multiple muscle groups in the body.Tizanidine (Zanaflex Capsules™), temporarily reduces spasticity byblocking nerve impulses. Baclofen acts on the central nervous system torelax muscles. It also decreases the rate of muscle spasms, pain,tightness and improves range of motion. Benzodiazepines (Valium® andKlonopin®) act on the central nervous system to relax muscles andtemporarily decrease spasticity. Dantrolene sodium (Dantrium®) actsdirectly on the muscle by blocking the signals that cause muscles tocontract. Dantrolene use can lessen muscle tone. Injections of botulinumtoxin (Botox® or Myobloc®) relax stiff muscles, but the shots onlytarget specific limbs or muscle groups affected by spasticity. In moreextreme cases, intrathecal baclofen or surgery is used to relievespasticity.

While the precise mechanisms responsible for the development ofspasticity are not fully understood, a role for reduction of inhibitionis most likely involved. For example, inhibitory interneurons areparticularly vulnerable to spinal cord ischemic injury, and are oftenselectively lost following spinal cord injury. This may contribute tothe changes in inhibitory circuits observed following spinal injury suchas reduction in reciprocal inhibition as well as attenuation ofinhibition normally mediated by segmental inhibitory interneurons thatsynapse directly onto motoneurons.

The treatment of spasticity is treated by grafting inhibitoryinterneurons (MGE cells) into the affected segments of the injuredspinal cord. The GABAergic cells derived from the median ganglioniceminence (MGE) will down-regulate the hyperactive local spinalcircuitry. In the developing brain, MGE cells gives rise to inhibitoryneurons, and this particular class of neuron has a remarkable ability tomigrate and integrate in adult brain tissue. MGE cells will integrate inthe spinal cord and produce factors that will inhibit local circuitactivity and reduce post-lesion spasticity. This strategy can also beused to relieve spasticity in a variety of other conditions includingmultiple sclerosis, stroke, and brain injury.

Example 5 Prophetic Generation of a Spinal Cord Injury in Mice

Genetically modified and wild-type mice are anesthetized with Avertinsupplemented with isoflurane or isoflurane only. The skin over themiddle of the back is shaved. The shaved area is disinfected withClinidine. All surgical tools are soaked overnight in Cidex prior totheir use. Lubricating ophthalmic ointment is placed in each eye.Animals are placed on a warming blanket to maintain temperature at 37°C. A dorsal midline incision, approximately 1 cm in length is made usinga scalpel blade. The spinous process and lamina of T9 are identified andremoved. A circular region of dura, approximately 2.4 mm in diameter, isexposed. At this point the animal is transferred to the spinal cordinjury device that is about 5 feet from the surgical area. Smallsurgical clamps are placed on a spine rostral and a spine caudal tolaminectomy site to stabilize the vertebral column. Thereafter, a 2-3 gweight is dropped 5.0 cm onto the exposed dura. This produces a moderatelevel of spinal cord injury. Immediately after injury, the animal isremoved from the injury device and returned to the surgical area. Asmall, sterile suture is placed in the paravertebral musculature to markthe site of injury. The skin is then closed with wound clips and theanimal recovered from the anesthesia. The entire surgical procedure iscompleted within 45 to 60 minute.

Assessment of Motor Function in Mice

Spinal cord injured mice are euthanized at 1 hour to 42 days afterinjury. In those animals that survive 42 days, locomotor ability isevaluated as follows:

Open Field Testing.

This involves testing animals at 3 days and weekly thereafter until timeof euthanasia at 42 days. Locomotor testing consists of evaluating howanimals locomote in an open field. This open field walking scoremeasures recovery of hindlimb movements in animals during free openfield locomotion as described by Basso et al. A score of 0 is given ifthere is no spontaneous movement, a score of 21 indicate normallocomotion. Plantar stepping with full weight support and completeforelimb-hindlimb coordination is reached when an animal shows a scoreof 14 points. We use a modified version of the BBB score if the sequenceof recovering motor features is not the same as described in theoriginal score. If this is observed, points for the single features areadded independently. For example, a mouse showing incomplete toeclearance, enhanced foot rotation and already a ‘tail-up’ position, oneadditional point are added to the score for the tail position.

The mice are tested preoperatively in an open field, which was an80×130-cm transparent plexiglass box, with walls of 30 cm and apasteboard covered non-slippery floor. In postoperative sessions twopeople, blinded to the treatments, will observe each animal for a periodof 4 min. Animals that exhibit coordinated movement, based upon openfield testing, are subjected to additional tests of motor function asfollows.

Grid Walking.

Deficits in descending motor control are examined by assessing theability of the animals to navigate across a 1 m long runway withirregularly assigned gaps (0.5-5 cm) between round metal bars. The bardistances are randomly changed from one testing session to the next. Theanimals are tested over a period of 5 days, beginning 1 to 2 weeks priorto euthanasia.

Crossing this runway requires that animals accurately place their limbson the bars. In baseline training and postoperative testing, everyanimal will cross the grid for at least three times. The number offootfalls (errors) are counted in each crossing and a mean error rate iscalculated. If an animal is not able to move the hindlimbs, a maximum of20 errors are given. The numbers of errors counted are also rated in anon-parametric grid walk score: 0-1 error is rated as 3 points, 2-5 as 2points, 6-9 as 1 point and 10-20 footfalls as 0 points.

Foot Placement.

Footprint placement analysis is modified from De Medinaceli et al. Theanimal's hind paws are inked, for example, with watercolor paint thatcan easily be washed off, and footprints are made on paper covering anarrow runway of 1 m length and 7 cm width as the animals traverse therunway. This ensures that the direction of each step is standardized inline. A series of at least eight sequential steps are used to determinethe mean values for each measurement of limb rotation, stride length andbase of support. The base of support are determined by measuring thecore to core distance of the central pads of the hind paws. The limbrotation are defined by the angle formed by the intersection of the linethrough the print of the third digit and the print representing themetatarsophalangeal joint and the line through the central pad parallelto the walking direction. Stride length are measured between the centralpads of two consecutive prints on each side.

To include animals with incomplete weight support in early postoperativetesting sessions, a 4-point scoring system is also used: 0 points isgiven for constant dorsal stepping or hindlimb dragging, i.e. nofootprint was visible; 1 point is counted if the animal had visible toeprints of at least three toes in at least three footprints; 2 points aregiven if the animal showed exo- or endo-rotation of the feet of morethan double values as compared to its own baseline values; 3 points arerecorded if the animal showed no signs of toe dragging but footrotation; 4 points are rated if the animal showed no signs of exo- orendo-rotation (less than twice the angle of the baseline values). Theseanimals are tested over a period of 5 days, beginning 1 to 2 weeks priorto euthanasia.

Beam Balance.

Animals are placed on a narrow beam, and the ability to maintain balanceand/or traverse the beam is evaluated. These animals are tested over aperiod of 5 days, beginning 1 to 2 weeks prior to euthanasia. The narrowbeat test is performed according to the descriptions of Hicks andD'Amato. Three types of beams are used as narrow pathways: a rectangular2.3 cm wide bean, a rectangular 1.2 cm wide beam and a round dowel with2.5 cm diameter. All beams are 1 m long and elevated 30 cm from theground. After training, normal rats are expected to be able to traversethe horizontal beams with less than three footfalls. When occasionallytheir feet slipped off the beam, the animals are retrieved andrepositioned precisely.

A scoring system is used to assess the ability of the animals totraverse the beams: 0 is counted as complete inability to walk on thebean (the animals fall down immediately), 0.5 is scored if the animalwas able to traverse half of the beam, 1 point is given for traversingthe whole length, 1.5 points when stepping with the hindlimbs ispartially possible, and 2 points is noted for normal weight support andaccurate foot placement. If the scores of all three beams are added, amaximum of 6 points can be reached.

Inclined Plane.

Animals are placed on a platform that can be raised to varying angles.The ability to maintain position at a given angle is determined. Theseanimals are tested over a period of 5 days, beginning 1 to 2 weeks priorto euthanasia. Animals are placed on an adjustable inclined planeconstructed as described (Rivlin and Tator, 1977). The slope isprogressively increased every 20 s noting the angle at which the mousecould not maintain its position for 5 s. The test is repeated twice foreach mouse and the average angle is recorded. In the inclined-planetest, recovery from motor disturbance is assessed before, and again at1, 7, 14, and 21 d after the injury. The maximum inclination of theplane on which the rats could maintain themselves for 5 sec withoutfalling is recorded.

Each of these tests described above takes less than 5 minutes. Thesevarious tests are designed such that if animals fall from the testingapparatus, they either land on padded flooring or the distance fallen issufficiently limited (less than 6 inches) that the animals are not beharmed.

Generation of a Spinal Cord Injury in Rats

MGE cells are implanted into the uninjured cord of rats to assess theirintegration into the local circuitry (n=10) and also into contused(n=10) and transected (n=10) spinal cords. Both contusion andtransection are studied in order to assess mild (contusion) and moderate(transection) levels of spasticity.

Rats are anesthetized with Avertin supplemented with isoflurane orisoflurane only. The skin over the middle of the back is shaved. Theshaved area is disinfected with Clinidine. All surgical tools are soakedovernight in Cidex prior to their use. Lubricating ophthalmic ointmentis placed in each eye. Animals are placed on a warming blanket tomaintain temperature at 37° C. A dorsal midline incision, approximately1 cm in length is made using a scalpel blade. The spinous process andlamina of T9 are identified and removed. A circular region of dura,approximately 2.4 mm in diameter, is exposed.

To produce a contusion injury, a circular region of dura, approximately3.0 mm in diameter, is exposed. At this point the animal is transferredto the spinal cord injury device that is about 5 feet from the surgicalarea. Small surgical clamps are placed on a spine rostral and a spinecaudal to laminectomy site to stabilize the vertebral column.Thereafter, a 10 g weight is dropped 5 cm onto the exposed dura. Thisproduces a moderate level of spinal cord injury. To produce atransection injury, the procedure to expose the cord is similar to thatfor the contusion injury except a surgical blade is used to completelytransect the spinal cord.

Immediately after contusion or transection injury, the animal is removedfrom the injury device and returned to the surgical area. A small,sterile suture is placed in the paravertebral musculature to mark thesite of injury. The skin is then closed with wound clips and the animalrecovered from the anesthesia. The entire surgical procedure iscompleted within 45 to 60 minutes.

Spinal Cord Implantation of MGE Cells in Rats

The first surgery produces either a contusion or transection spinal cordinjury as described. Spasticity is initially seen at seven days afterinjury. A second surgery on day 7 is used to inject MGE cells into theinjured spinal cord. The delay in MGE implantation allows the ability tomeasure reductions in spasticity over time.

Seven days after either transection or contusion, animal arere-anesthetized with isoflurane, the exposed cord is visualized and twoadditional laminectomies are made caudal to the original laminectomy.Using a stereotaxic device, MGE cells, are injected into each of theventral horns. An uninjured control group is also be injected withcells. A sterile suture is positioned in the paravertebral musculatureto mark the surgical site. The skin is then closed with wound clips andthe animal is recovered from the anesthesia. This transplantationprocedure is completed in approximately 60 minutes. Spinal cord injuredand control rats are anesthetized and perfused with fixative at 1 to 6weeks after transplantation of cells.

Assessment of Motor Function in Rats

After recovery from surgery, these animals may be assessed for function1-2 times weekly:

Locomotor Assessment.

Locomotor testing consists of evaluating how animals locomote in an openfield. One day postinjury (p.i.) and 1 to 2 times weekly thereafter,behavioral analysis is performed by two observers blinded to thetreatments, using a battery of tests to rate open-field locomotion bythe Basso-Beattie-Bresnahan (BBB) scale. This open field walking scoremeasures recovery of hindlimb movements in rats during free open fieldlocomotion as described by Basso et al. A score of 0 is given if thereis no spontaneous movement, a score of 21 indicate normal locomotion.Plantar stepping with full weight support and complete forelimb-hindlimbcoordination is reached when an animal shows a score of 14 points. Wewill use a modified version of the BBB score if the sequence ofrecovering motor features is not the same as described in the originalscore. If this is observed, points for the single features are addedindependently. For example, a rat showing incomplete toe clearance,enhanced foot rotation and already a ‘tail-up’ position, one additionalpoint are added to the score for the tail position.

The rats are tested preoperatively in an open field, which was an80×130-cm transparent plexiglass box, with walls of 30 cm and apasteboard covered non-slippery floor. In postoperative sessions twopeople, blinded to the treatments, will observe each animal for a periodof 4 min. Animals that exhibit coordinated movement, based upon openfield testing, are subjected to additional tests of motor function asfollows.

Grid Walking.

Deficits in descending motor control are examined by assessing theability of the animals to navigate across a 1 m long runway withirregularly assigned gaps (0.5-5 cm) between round metal bars. The bardistances are randomly changed from one testing session to the next. Theanimals are tested 1 to 2 times weekly postinjury. Crossing this runwayrequires that animals accurately place their limbs on the bars. Inbaseline training and postoperative testing, every animal will cross thegrid for at least three times. The number of footfalls (errors) arecounted in each crossing and a mean error rate is calculated. If ananimal is not able to move the hindlimbs, a maximum of 20 errors aregiven. The numbers of errors counted are also rated in a non-parametricgrid walk score: 0-1 error is rated as 3 points, 2-5 as 2 points, 6-9 as1 point and 10-20 footfalls as 0 points.

Foot Placement.

Footprint placement analysis is modified from De Medinaceli et al. Theanimal's hind paws are inked, for example, with watercolor paint thatcan easily be washed off, and footprints are made on paper covering anarrow runway of 1 m length and 7 cm width as the animals traverse therunway. This ensures that the direction of each step is standardized inline. A series of at least eight sequential steps are used to determinethe mean values for each measurement of limb rotation, stride length andbase of support. The base of support are determined by measuring thecore to core distance of the central pads of the hind paws. The limbrotation are defined by the angle formed by the intersection of the linethrough the print of the third digit and the print representing themetatarsophalangeal joint and the line through the central pad parallelto the walking direction. Stride length are measured between the centralpads of two consecutive prints on each side.

To include animals with incomplete weight support in early postoperativetesting sessions, a 4-point scoring system is also used: 0 points isgiven for constant dorsal stepping or hindlimb dragging, i.e. nofootprint was visible; 1 point is counted if the animal had visible toeprints of at least three toes in at least three footprints; 2 points aregiven if the animal showed exo- or endo-rotation of the feet of morethan double values as compared to its own baseline values; 3 points arerecorded if the animal showed no signs of toe dragging but footrotation; 4 points are rated if the animal showed no signs of exo- orendo-rotation (less than twice the angle of the baseline values).

Beam Balance.

Animals are placed on a narrow beam, and the ability to maintain balanceand/or traverse the beam is evaluated. The narrow beat test is performedaccording to the descriptions of Hicks and D'Amato. Three types of beamsare used as narrow pathways: a rectangular 2.3 cm wide bean, arectangular 1.2 cm wide beam and Three types of beams are used as narrowpathways: a rectangular 2.3 cm wide bean, a rectangular 1.2 cm wide beamand a round dowel with 2.5 cm diameter. All beams are 1 m long andelevated 30 cm from the ground. After training, normal rats are expectedto be able to traverse the horizontal beams with less than threefootfalls. When occasionally their feet slipped off the beam, theanimals are retrieved and repositioned precisely.

A scoring system is used to assess the ability of the animals totraverse the beams: 0 is counted as complete inability to walk on thebean (the animals fell down immediately), 0.5 is scored if the animalwas able to traverse half of the beam, 1 point is given for traversingthe whole length, 1.5 points when stepping with the hindlimbs ispartially possible, and 2 points is noted for normal weight support andaccurate foot placement. If the scores of all three beams are added, amaximum of 6 points can be reached.

Inclined Plane.

Animals are placed on a platform that can be raised to varying angles.The ability to maintain position at a given angle is determined.

Animals are placed on an adjustable inclined plane constructed asdescribed (Rivlin and Tator, 1977). The slope is progressively increasedevery 20 s noting the angle at which the mouse could not maintain itsposition for 5 s. The test is repeated twice for each mouse and theaverage angle is recorded. In the inclined-plane test, recovery frommotor disturbance is assessed before, and again at 1, 7, 14, and 21 dafter the injury. The maximum inclination of the plane on which the ratscould maintain themselves for 5 sec without falling is recorded.

Each of these tests described above takes less than 5 minutes. Thesevarious tests are designed such that if animals fall from the testingapparatus, they either land on padded flooring or the distance fallen issufficiently limited (less than 6 inches) that the animals are notharmed.

Transplantation of MGE progenitor cells can be used in methods of thepresent disclosure reduce spasticity (e.g., as seen in multiplesclerosis, stroke, and brain injury) by at least 10% compared tocontrols, or the individual's previously untreated stated. In someembodiments, transplantation of MGE progenitor cells reduces spasticityby at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared tocontrols, or the individual's previously untreated stated. In someembodiments, transplantation of MGE progenitor cells reduces the needfor medication by at least 10% compared to controls, or the individual'spreviously untreated stated. In some embodiments, transplantation of MGEprogenitor cells reduces the need for medication by at least 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to controls, or theindividual's previously untreated stated. In some embodiments,transplantation of MGE progenitor cells obviates the need forintrathecal medication or surgery.

In Vitro Expansion of MGE Progenitor Cells (Prophetic Example)

The practical and ethical problems associated with embryonic and fetaltissue transplantation is overcome through the use of in vitro expansionof a small amount of embryonic or fetal CNS tissue as floating cellaggregates called neurospheres using the technique developed byReynolds, B. A., and Weiss, S., Generation of neurons and astrocytesfrom isolated cells of adult mammalian central nervous system. Science255: 1707-1710, 1992.

Primary Cultures and Passaging Procedures

Adult pregnant rats are anesthetized with ketamine (90 mg/Kg) andxylazine (7 mg/Kg). Fetuses are removed from the uterus at E14.5 andbrains dissected under a microscope. The MGE is dissected from theforebrain in oxygenated artificial cerebrospinal fluid (aCSF, in mm:NaCl, 125; KCl, 2.5; MgCl2, 1; CaCl2, 2; NaPO4, 1.25; NaHCO3, 25; andglucose, 25; Sigma). Twelve to 15 MGEs are dissected and prepared asdescribed in Reynolds and Weiss. The culture medium is DMEM/F12supplemented with the hormone mixture used by Reynolds and Weiss alongwith human recombinant fibroblast growth factor-2 (FGF-2; 20 ng/ml;Prepro Tech, Inc., Rocky Hill, N.J.). Fresh FGF-2 is added every 2 days.Cells are passages once per week. The passage of cells obtained from onepregnant rat (12-15 fetuses) three times will produce enough immatureneural progenitor cells for the transplantation of at least 450 rats.Thus, the in vitro expansion of embryonic or fetus-derived MGEprogenitor cells using the above methodology or other methodology knownto one of skill in the art is a useful source for transplantablematerial with which to treat neurological conditions, disease, orinjury, including Parkinson's disease, epilepsy, and spinal cord injury.

Generation of an Immortalized Cell Line that is Substantially Similar toMGE Cells Isolated from E13.5 Mouse Fetus (Prophetic Example)

Normal human somatic cells have a defined life span and enter senescenceafter a limited number of cell divisions. At senescence, cells areviable but no longer divide. This limitation on cell proliferationrepresents an obstacle in the scale up and production of largequantities of MGE progenitor cells required for transplantation. Theestablishment of permanent cell lines will circumvent this problem. Manyimmortalized tumor cell lines are known and normal human somatic cellscan be transformed in vitro with oncogenes to establish permanent celllines. While valuable for research, transformed cells derived fromnormal human cells are unacceptable for therapeutic use owing to theiralterations in cell cycle, loss of contact inhibition, aneuploidy,spontaneous hypermutability and other characteristics associated withcancerous cells.

Fortunately, the recognition that replicative senescence is controlledby the shortening of the telomeres allows for the production ofimmortalized cell lines through methodologies that modulate or controlthe regulation of telemerase. One of the number of ways known in the artto immortalize cells is by the forced expression of exogenous hTERT innormal human cells. This produces telomerase activity and prevent theerosion of telomeres and resultant senescence. MGE progenitor cellsimmortalized with hTERT have normal cell cycle controls, functional p53and pRB checkpoints, are contact inhibited, are anchorage dependent,require growth factors for proliferation and possess a normal karyotype.Thus MGE progenitor cells that express exogenous hTERT have extendedlife spans that allow for the production of useful quantities of MGEprogenitor cells for transplantation.

Methods and Materials

Telomerase is introduced into MGE progenitors using the techniques ofOuellette, M. M. et al. The establishment of telomerase-immortalizedcell lines representing human chromosome instability syndromes. HumanMolecular Genetics, 9:403-411, 2000. Briefly, retroviral supernatants isobtained from PA317 packaging cells stably expressing hTERT cloned intothe pBabepuro vector. Cells are infected and then selected for 2 weeksusing puromycin at 750 ng/ml.

Cells are grown at 37° C. in 5% CO₂ Neurobasal™ Medium (Invitrogen,Purchase, N.Y.) and Gentamicin (25 μg/ml). For hygromycin B sensitivity,cells were grown at various doses for 7 days, stained for viability andcounted.

Telomerase activity is determined using the TRAP assay (TRAPeze kit,Intergen, Purchase, N.Y.). PCR products are electrophoresed on 10%polyacrylamide gels and quantified using the Phosphorimaging system andIMAGEQUANT (Molecular Dynamics, Amersham Pharmacia Biotech, Piscataway,N.J.). Quantitation of telomerase activity is done by determining theratio of the 36 bp internal standard to the telomerase ladder.

Total genomic DNA is isolated as described in Ouellette, M. M., et al.Telomerase activity does not always imply telomere maintenance. Biochem.Biophys. Res. Commun., 254:795-803, 1999. The DNA is digested with abattery of six enzymes (HinfI, RsaI, CfoI, AluI, HaeIII, MspI) andresolved on a 1% agarose gel. The gel is denatured and dried,neutralized and the signal detected in situ using a telomeric probeend-labeled with [γ-³²P]ATP.

The immortalized MGE progenitor cell line is compared with uninfectedMGE progenitor cells to demonstrate: (i) the presence of telomeraseactivity; (ii) the capacity to maintain extended telomere size; and(iii) the ability to grow beyond the number of population doublings ofuninfected control MGE progenitor cells. Using the telomere repeatamplification protocol (TRAP) assay, telomerase activity should be foundto be absent from all uninfected samples, but present in samples thatare infected with the hTERT vector. Preferably, the level of activitydetected in the infected cells should be comparable with that detectedin a human lung cancer cell line, H1299, with telomere size larger inthe immortalized MGE progenitor cells than in their uninfectedcounterpart, indicating the exogenous telomerase activity is able toelongate telomeres. All samples are continuously passaged to determinecellular life span. Uninfected samples would be expected to entersenescence after approximately 60 population doublings, and thus thepresence of exogenous telomerase preferably should extend the life spanof MGE progenitor cells by at least three-fold compared to theuninfected cultures.

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While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method for the treatment of a mammal having a neurologicalcondition, disease, or injury comprising: increasing the number offunctional GABAergic interneurons at or near the site of theneurological disease, injury, or condition, wherein said functionalGABAergic interneurons functionally integrate with endogenous neuronsand restore balance to neuronal circuitry that is dysregulated inneurological conditions, diseases, or injuries.
 2. The method of claim1, wherein the increase in said functional GABAergic interneurons is bytransplantation comprising the injection of MGE (medial ganglioniceminence) precursor cells.
 3. The method of claim 2, wherein the MGEprecursor cells are able to migrate at least 0.5 mm from thetransplantation site.
 4. The method of claim 1, wherein the neurologicalcondition, disease, or injury is a degenerative disease, geneticdisease, acute injury, or chronic injury.
 5. The method of claim 4,wherein the neurological condition, disease, or injury comprisesParkinson's disease, epilepsy, spasticity, multiple sclerosis, stroke,spinal cord injury, brain injury, or chronic pain disorders.
 6. Themethod of claim 3, wherein the MGE precursor cells express a therapeuticprotein or peptide, or neurotransmitter.
 7. The method of claim 6,wherein the therapeutic protein or peptide comprises a neurotrophin, aneuropoietic cytokine, a fibroblast growth factors (e.g., acidic andbasic FGF), an inhibitory growth factor, or a cytokine useful in thetreatment of infectious disease, brain tumors, or brain metastases. 8.The method of claim 4, wherein the neurological condition is epilepsy,wherein transplantation of MGE precursor cells result in at least a 10%reduction in spontaneous electrographic seizure activity.
 9. The methodof claim 4, wherein the neurological condition is epilepsy, whereintransplantation of MGE precursor cells result in at least a 10%reduction in seizure duration.
 10. The method of claim 4, wherein theneurological condition is epilepsy, wherein transplantation of MGEprecursor cells result in at least a 10% reduction in seizure frequency.11. The method of claim 4, wherein the neurological condition isepilepsy, wherein transplantation of MGE precursor cells result in atleast a 10% reduction in required antiepileptic drug use.
 12. The methodof claim 4, wherein the neurological disease is Parkinson's disease,wherein transplantation of MGE precursor cells result in at least a 10%reduction in required antiParkinsonian drug use.
 13. The method of claim4, wherein the neurological disease is Parkinson's disease, whereintransplantation of MGE precursor cells result in at least a 10%reduction in tremor at rest, rigidity, akinesia, bradykinesia, posturalinstability, flexed posture and/or freezing.
 14. The method of claim 4,wherein the neurological disease is Parkinson's disease, wherein the MGEcells transplanted into the striatum survive for at least 6 months. 15.The method of claim 4, wherein the neurological condition is spasticity,wherein transplantation of MGE precursor cells obviates the need forintrathecal medication or surgery.
 16. The method of claim 4, whereinthe neurological condition is spasticity, wherein transplantation of MGEprecursor cells result in at least a 10% reduction in requiredantispasmodic drug use.
 17. The method of claim 2, wherein the MGEprecursor cells are injected into the striatum, basal ganglia, dorsalganglia, ventral horn, or lumbar theca.
 18. The method as in claim 2,wherein the mammal does not require immunosuppressive therapy followingtransplantation.
 19. A composition comprising MGE precursor cells and apharmaceutically acceptable excipient.
 20. The composition of claim 19,wherein the MGE precursor cells express a therapeutic protein orpeptide.